JOURNAL OF VIROLOGY, July 2005, p. 9228–9235
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 14
CD8?T-Lymphocyte Response to Major Immunodominant Epitopes
after Vaginal Exposure to Simian Immunodeficiency
Virus: Too Late and Too Little
Matthew R. Reynolds,1,2Eva Rakasz,1Pamela J. Skinner,3Cara White,3† Kristina Abel,4,5,6
Zhong-Min Ma,4,5,6Lara Compton,4,5,6Gnankang Napoe ´,1Nancy Wilson,1
Christopher J. Miller,4,5,6Ashley Haase,7*
and David I. Watkins1,2*
Wisconsin Primate Research Center1and Department of Pathology and Laboratory Medicine,2University of Wisconsin, Madison,
Wisconsin 53715; Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota 551083;
Center for Comparative Medicine, California National Primate Research Center,4Department of Pathology, Microbiology,
and Immunology, School of Veterinary Medicine,5and Department of Infectious Disease, School of Medicine,6
University of California, Davis, California 95616; and Department of Microbiology, University of Minnesota
Medical School, Minneapolis, Minnesota 554557
Received 6 December 2004/Accepted 2 March 2005
In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and
simian immunodeficiency virus (SIV), virus-specific CD8?T-lymphocyte responses partially control but do not
eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4?T
lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure
to clear infection and prevent CD4?T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal
transmission. We examined the relationship between the timing and magnitude of the CD8?T-lymphocyte
response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain
infection is not because the female reproductive tract is a poor inductive site. We documented robust responses
in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we
also documented a modest response in the draining genital and peripheral lymph nodes, the response at these
sites also lagged behind peak virus production in these LT compartments. Third, we found that the response
in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of
CD4?T lymphocytes in the GALT. Thus, the virus-specific CD8?T-lymphocyte response is “too late and too
little” to clear infection and prevent CD4?T-lymphocyte loss. However, the robust response in female
reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8?
T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of
In the natural history of sexual transmission of human im-
munodeficiency virus type 1 (HIV-1) and simian immunodefi-
ciency virus (SIV), systemic infection is established throughout
the lymphatic tissues (LTs) within the first weeks after expo-
sure. Viral production and viremia peak at this time and then
decline, temporally coincident with the development of virus-
specific CD8?T-lymphocyte responses (5, 14, 16, 24). How-
ever, in contrast to many other viral infections, the immune
system is unable to completely clear infection. Depletion of
CD4?T lymphocytes, which is particularly severe in the gut-
associated lymphoid tissue (GALT) (6, 19, 29, 33), is demon-
strable in this acute stage in both HIV-1 and SIV infections
and will generally progress in all LT compartments to levels
where AIDS-associated opportunistic infections and cancers
are manifest unless infection is controlled with antiretroviral
Understanding why HIV and SIV win this struggle with host
defenses might provide important insights into designing an
effective HIV vaccine. To that end, we investigated three hy-
pothetical explanations for the failure to prevent infection and
CD4?T-lymphocyte loss following vaginal exposure. (i) The
female reproductive tract is a poor inductive site to mount a
virus-specific immune response and cannot thus prevent dis-
semination and establishment of systemic infection. (ii) The
host mounts a robust response but not in enough time to
prevent widespread systemic dissemination and establishment
of a persistent infection in LTs. (iii) The response is of insuf-
ficient magnitude relative to the number of infected cells to
fully contain infection.
We investigated these hypotheses with the SIV rhesus ma-
caque model of vaginal transmission of HIV-1 by determining
the relationship between the immune response in tissue com-
partments and the kinetics and magnitude of infection in those
compartments described in the accompanying report. We fo-
cused on the virus-specific CD8?T-lymphocyte response be-
* Corresponding author. Mailing address for Ashley Haase: Depart-
ment of Microbiology, University of Minnesota, MMC 196, 420 Del-
aware St. S.E., Minneapolis, MN 55455. Phone: (612) 624-4442. Fax:
(612) 626-0623. E-mail: firstname.lastname@example.org. Mailing address for
David I. Watkins: Wisconsin Primate Research Center, 1220 Capitol
Ct., Madison, WI 53715. Phone: (608) 265-3380. Fax: (608) 263-4031.
† Present address: Rio Grande City High School, Rio Grande City,
cause of several lines of evidence that suggest that these re-
sponses partially control viral replication during acute
infection. (i) Antigen-specific T lymphocytes are temporally
associated with a reduction in plasma viremia (5, 14, 16, 24).
(ii) CD8 depletion results in high-level viral replication (18,
25). (iii) The emergence of escape variants indicates selective
pressure exerted by the CD8?T lymphocytes (3, 22). We
specifically investigated the Mamu-A*01-restricted immuno-
dominant epitopes, Gag181-189CM9 and Tat28-35SL8, because
the response to these two epitopes accounts for the majority of
detectable CD8?T-lymphocyte responses in Mamu-A*01-pos-
itive macaques (21) and because Gag CM9-specific responses
following intravenous transmission have been documented in
the mucosal and lymphoid tissue compartments which are rel-
evant to understanding the reasons why immune defenses fail
to fully protect animals following intravaginal exposure. Vea-
zey et al., for example, detected Gag CM9-specific CD8?T
lymphocytes in the blood and the intestinal mucosa at 2 and 3
weeks postintravenous infection at similar frequencies (30, 32).
During the chronic phase of infection, virus-specific CD8?T
lymphocytes were detected at similar levels in peripheral blood
mononuclear cells (PBMC) and secondary lymphoid tissues
(16), and tetramer-staining lymphocytes were found at compa-
rable, or higher, frequencies in the vagina and intestinal mu-
cosa (26, 28, 32).
Here, we show first that CD8?lymphocytes in female re-
productive tissues do respond to infection at the portal of
entry, but there is a surprisingly long lag between CD8?T-
lymphocyte responses to the immunodominant SIV epitopes
and peak levels of infection at the portal of entry and all LT
compartments. Second, the magnitude of the virus-specific
CD8?T-lymphocyte responses, while relatively robust at the
portal of entry, was modest in LTs generally and low or unde-
tectable in the GALT. The overall disparity in the timing and
magnitude of immunodominant response with respect to prop-
agation and dissemination of infection is thus a major factor in
the failure to clear infection and prevent CD4?T-cell loss,
particularly preferential depletion of CD4?T lymphocytes in
MATERIALS AND METHODS
Monkeys, SIV inoculum, and infection status. As described in the accompa-
nying paper by Miller et al. (19a), eight Mamu-A*01 rhesus monkeys became
systemically infected after intravaginal inoculation.
Isolation of PBMC. PBMC were isolated from heparin- or EDTA-treated
blood samples using Lymphocyte Separation medium (ICN Biomedicals, Au-
rora, OH). PBMC not used in fresh assays were frozen in 10% dimethyl sulfoxide
(Sigma, St. Louis, MO)–90% fetal bovine serum (Gemini BioProducts, Calaba-
sas, Calif.) and stored in liquid nitrogen for future analyses.
Lymphoid tissue collection and lymphocyte isolation. At necropsy, blood,
intestine, spleen, and peripheral and genital lymph node (LN) samples were
collected. In addition, cell suspensions were prepared as previously described
(17) from whole blood, spleen, and peripheral and genital lymph nodes for
gamma interferon (IFN-?) enzyme-linked immunospot assay and fluorescence-
activated cell sorter (FACS) analysis by gentle dissection of lymph nodes with
scalpels in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum (Gemini BioProducts, Calabasas, CA) (complete RPMI) and pas-
sage of the cell homogenate through a cell strainer (Fisher, Pittsburgh, PA). The
cells were washed twice by centrifugation for 10 min at 1,600 rpm. Spleen tissue
samples were cut into small pieces and homogenized with a syringe plunger. The
homogenate was passed through a cell strainer. Splenic lymphocytes were iso-
lated by gradient centrifugation with Lymphocyte Separation medium from ICN
Biomedicals (Aurora, OH), followed by two washes with complete RPMI. PBMC
were isolated from whole blood with Lymphocyte Separation medium (ICN).
Isolation of lymphocytes from the cervix and vaginal mucosa. At necropsy,
fresh cervix and vagina were collected in complete RPMI medium containing
10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml strep-
tomycin (Sigma, Inc., St Louis, MO,) and 1% amphotericin B (Sigma). Lympho-
cytes were isolated as previously described (17) from genital mucosal tissues by
cutting the tissues into small pieces (maximum size, 10 by 10 by 5 mm) and
placing them in 1.2 U/ml Dispase II (Boehringer Mannheim GmbH, Germany)
in complete RPMI medium (3 ml per piece). The tissue-dispase II mixture was
incubated in a water bath shaker (New Brunswick Scientific, Inc., Edison, NJ) for
2 h at 37°C and 200 rpm. After being rinsed, the epithelial layer was removed
from the underlying tissue with forceps and scalpel. The subepithelial tissues
were cut into 2- by 5-mm slices (each, approximately 2 mm thick) and incubated
in cell release medium. Cell release medium is complete RPMI 1640 containing
25 mM HEPES (Sigma), 5 ? 10?3M ?-mercaptoethanol (Sigma), 0.5 mg/ml
collagenase (Sigma), 0.1 mg/ml DNase (Sigma), and 0.02 mg/ml ciprofloxacin-
HCl. The tissue was incubated for 3 to 4 h in a shaking water bath at 37°C and
100 rpm. To obtain cell suspensions from the lamina propria, 40 ml of the
submucosal tissue suspension was shaken vigorously in a tube for 90 s. The
resulting cell suspension was then passed through a 100-mesh sterile stainless
steel sieve to remove larger pieces of debris and centrifuged at 530 ? g for 10
min. Mononuclear cells were subsequently isolated on a discontinuous Percoll
(Pharmacia) gradient. Cells were resuspended in 40% Percoll in RPMI medium,
layered onto 75% Percoll in phosphate-buffered saline (PBS), and centrifuged at
530 ? g for 30 min using the no-brake setting. Cell viability, measured by trypan
blue exclusion, was typically ?95%.
Lymphocyte isolation from uterus. After being extensively washed in PBS, the
uterus was cut into 4- to 6-mm2pieces. The tissues were digested in R10 (RPMI
1640 supplemented with 10% fetal calf serum [FCS], 2 mM L-glutamine, 25 mM
HEPES buffer, 50 ?g/ml streptomycin, 50 U/ml penicillin) containing type IV
collagenase (0.5 mg/ml) (Sigma) and DNase type IV (100 U/ml) (Sigma) for 2 h
at 37°C. The supernatant was collected, and lymphocytes were enriched by
density gradient centrifugation using a 90% to 40% isotonic Percoll gradient at
1,200 rpm for 30 min at room temperature. The lymphocytes were collected from
the 90% to 40% Percoll interface. Cells were washed in R10.
Isolation of lymphocytes from GALT. Sections of colon and jejunum, 6- to 8-in
long, were cut into approximately 5-mm2pieces and incubated in complete
Hank’s balanced salt solution (Sigma, St. Louis, MO) supplemented with 10%
FCS, 25 mM HEPES buffer, and 1 mM EDTA at 37°C for 90 min. Supernatant,
containing intraepithelial lymphocytes (IEL), was collected and run over glass
wool loosely packed into a 30-ml syringe to remove large particulates.
Flowthrough was washed twice with 30 ml of R10. To release the lamina propria
lymphocytes (LPL), the remaining tissue was cut into small pieces of ?1 mm2
each and incubated for 90 min at 37°C in R10 containing type IV collagenase (15
U/ml) (Sigma, St. Louis, MO) and DNase type IV (100 U/ml) (Sigma, St. Louis,
MO). Supernatant was collected, run over loosely packed glass wool, and washed
twice in 30 ml of R10. Both IEL and LPL were enriched from epithelial cells by
centrifuging over a 90% to 40% isotonic Percoll gradient. Lymphocytes were
collected from the 90% to 40% Percoll interface.
Intracellular cytokine staining of fresh lymphocytes. Intracellular cytokine
(ICS) assays were performed as previously described (12). Briefly, 5 ? 105to 1
? 106freshly isolated lymphocytes were incubated with individual peptides at a
concentration of 5 ?g/ml along with 0.5 ?g anti-CD28 (clone L293; BD Bio-
sciences, San Diego, CA) and 0.5 ?g anti-CD49d (clone 9F10; BD Pharmingen,
San Diego, CA) for optimal costimulation in a total volume of 200 ?l R-10.
Staphylococcal enterotoxin B (10 ?g/ml; Sigma, St. Louis, MO) was used as a
positive control while R10, anti-CD28, and anti-CD49d alone were used as a
negative control. After incubation for 1.5 h at 37°C 10 ?g/ml of brefeldin A
(Sigma, St. Louis, MO) was added to inhibit secretion of cytokines. The cells
were further incubated for 5 h at 37°C. Cells were washed twice with FACS buffer
(PBS plus 2% FCS) and stained with anti-CD8?-PerCP (clone SK1; Becton
Dickinson) and anti-CD4-APC (clone SK3; Becton Dickinson) in 100 ?l FACS
buffer for 40 min at room temperature. After two washes, the cells were fixed
with 2% paraformaldehyde (PFA)–PBS solution overnight at 4°C. After being
washed twice with permeabilization buffer (0.1% saponin in FACS buffer) the
cells were stained with anti-human IFN-?–fluorescein isothiocyanate (FITC)
monoclonal antibody (MAb) (clone 4S.B3; Pharmingen) and anti-human tumor
necrosis factor alpha (TNF-?)–PE MAb (clone Mab11; Pharmingen) and incu-
bated for 50 min at room temperature. Afterwards, the cells were washed twice
with permeabilization buffer and fixed with 2% PFA–PBS. A total of 10,000 to
200,000 lymphocyte-gated events were acquired on a FACSCalibur flow cytom-
VOL. 79, 2005COMPARTMENTAL SIV IMMUNODOMINANT CD8?T-CELL RESPONSE9229
eter (Becton Dickinson) and analyzed with FlowJo software (Treestar). All
values are reported after subtraction of the background level staining.
Tetramer staining and FACS. From 5 ? 105to 1 ? 106freshly isolated
lymphocytes were suspended in a 100-?l volume of R10 and stained for 1 h at
37°C with Mamu-A*01/peptide tetramer labeled with phycoerythrin or antigen-
presenting cell. After a 1-h incubation, the lymphocytes were stained for surface
markers using anti-human CD3ε-FITC (SP34; Pharmingen) and anti-CD8?-
PerCP (clone SK1; Becton Dickinson) and incubated for an additional 40 min at
room temperature. The cells were washed twice with FACS buffer and fixed with
2% PFA–PBS. Sample data was acquired using a FACSCalibur flow cytometer
(Becton Dickinson) and analyzed using FlowJo software (Treestar). Background
staining of CD3?/CD8?lymphocytes in all tissues was typically ?0.1%.
In situ tetramer (IST) staining. Biotinylated Mamu-A*01/?2m/peptide mole-
cules were produced with Gag (CTPYDINQM), Tat (TTPESANL), or irrelevant
(FLPSDYFPSV) peptides at the National Institute of Allergy and Infectious
Diseases tetramer facility. Tetramers were generated by adding six aliquots of
FITC-labeled ExtraAvidin (Sigma) to biotinylated Mamu-A*01/?2m/peptide
monomers over the course of 8 h to a final molar ratio of 4.5:1. IST staining was
performed essentially as previously described (27). Fresh lymph nodes, spleen,
uterus, and vaginal and cervical tissues were shipped on ice overnight in PBS or
PBS with 100 ?g/ml heparin (PBS-H). Tissues were cut into 0.5-cm pieces and
embedded in 4% low-melt agarose. Tissue blocks were placed in a vibratome
bath containing 0 to 4°C PBS-H, and 200-mm-thick sections were generated.
Fresh sections were stained free floating in 1 ml of solution with four sections per
well in 24-well tissue culture plates. Incubations were carried out at 4°C on a
rocking platform. Tetramers were added at a concentration of 0.5 mg/ml with 2%
normal goat serum (NGS) and 0.5 mg/ml mouse anti-human CD8 antibodies
(Dako clone DK25) and incubated overnight. Sections were washed with chilled
PBS-H and then fixed with 4% paraformaldehyde for 2 h at room temperature.
Sections were again washed with PBS-H, incubated with rabbit anti-FITC anti-
bodies (BioDesign) diluted 1:10,000 in PBS-H with 2% NGS, and incubated for
1 to 3 days. Sections were washed three times with PBS-H for at least 20 min and
then incubated with Cy3-conjugated goat anti-rabbit antibodies and Alexa 488-
conjugated goat anti-mouse antibodies (Jackson ImmunoResearch), both diluted
1:1,000 in PBS-H with 2% NGS, for 1 to 3 days. Finally, sections were washed
three times for at least 20 min in PBS-H, postfixed with 4% paraformaldehyde
for 1 h, and then mounted on slides with warmed glycerol gelatin (Sigma)
containing 4 mg/ml n-propyl galate. Stained sections were analyzed using a
Bio-Rad 1000 confocal microscope.
CD8?T-lymphocyte responses to immunodominant SIV
epitopes lag behind peak levels of virus production at the
portal of entry and LTs. In the accompanying report, we show
that infection is established beyond the portal of entry in the
proximal and distal LTs by the end of the first week postintra-
vaginal inoculation (PI). We found that viral replication
peaked in all tissues and blood between days 10 and 14 PI and
then declined by an order of magnitude or more by day 28 PI,
the last time point examined.
Here, we investigated the relationship between propagation
and spread of infection and the development of SIV-specific
CD8?T-lymphocyte responses to the Mamu-A*01-restricted
immunodominant epitopes in Gag (Gag CM9) and Tat (Tat
SL8) that comprise the majority of the detectable virus-specific
CD8?T-lymphocyte responses during acute infection of
Mamu-A*01?macaques (21). To make optimal use of Mamu-
A*01?macaques, we first bracketed the times virus-specific
cellular immune responses were detectable in Mamu-A*01?
macaques by an optimized enzyme-linked immunospot assay
shown previously to be comparable in sensitivity to flow cyto-
metric assays (23). We did not detect significant cellular im-
mune responses until day 14 PI and then in only one of the two
animals. By 21 days PI, virus-specific cellular immune re-
sponses were detectable in the reproductive and secondary
lymphoid tissues (not shown). Therefore, we focused the de-
tailed compartmental analysis in Mamu-A*01?macaques at 13
to 28 days PI, spanning the peak through decay stages of
declining viral loads and productively infected cells (19a).
We determined the location and magnitude of epitope-spe-
cific T lymphocytes by collecting tissue and isolating lympho-
cytes from the female reproductive tract (vagina, cervix, and
uterus), the colon and jejunum, the proximal lymphoid tissues
draining the site of inoculation (genital and inguinal lymph
nodes), the distal lymphoid tissues (mesenteric lymph nodes,
axillary lymph nodes, and spleen) and the blood. CD8?T-
lymphocyte responses against Gag CM9, Tat SL8, and two
previously reported Mamu-A*01 restricted subdominant re-
sponses, Env233–241CL9 and Env620–628TL9 (10), were detected
by in situ staining and FACS analysis using Mamu-A*01/pep-
tide tetrameric complexes. Peptide-specific cytokine secretion
was monitored by ICS using antibodies against IFN-? and
TNF-?. We present only data for responses against Gag CM9
and Tat SL8, because measurable responses to the Env
epitopes were not detectable. Additionally, both IEL and LPL
were isolated from the colon and jejunum, but little or no
difference in the frequency of epitope-specific CD8?T-lym-
phocyte responses was detected between these compartments.
We therefore present only the responses from the LPL.
At 13 to 14 days PI, 4 days after the peak levels of virus in
female reproductive tissues (19a), we detected low but signif-
icant responses against the Gag CM9 and Tat SL8 epitopes in
the reproductive tract and in blood, but not elsewhere (Fig. 1)
in one of two animals. At days 20 to 21 PI, we detected Gag
CM9- and Tat SL8-specific responses at different levels in the
female reproductive tract tissues and lymphatic tissue compart-
ments in the three animals tested (Fig. 2 and Fig. 3A). The
tissues of the female reproductive tract consistently had high
frequencies of Gag CM9- and Tat SL8-specific CD8?T lym-
phocytes (range, 1 to 5%, with a high of 24%). In contrast to
this relatively robust response at the site of initial exposure,
there were only modest responses in the draining and distal
LNs and very low or undetectable responses in the GALT (Fig.
3A). Similarly, at days 27 to 28, the frequencies of tetramer-
FIG. 1. Low frequency of CD8?T-lymphocyte responses at 14 days
postintravaginal infection. The frequency of tetramer binding CD3?/
CD8?lymphocytes isolated from different lymphoid tissues is shown.
9230REYNOLDS ET AL. J. VIROL.
positive cells were highest in the reproductive tissues, followed
by the draining and/or distal LNs, and relatively low in the
colon and jejunum (Fig. 3B). IST staining of lymph nodes,
spleen, vagina, and cervix from each animal similarly revealed
Gag CM9- and Tat SL8-specific CD8?lymphocytes in repro-
ductive and lymphoid tissues at 20 to 21 and 27 to 28 but not
13 to 14 days PI (Fig. 4). Thus, particularly in GALT, the level
of immunodominant response not only lagged well behind the
peak of viral replication but was also the lowest of any LT
We found no reproducible difference in the distribution pat-
tern of the CD8?T-lymphocyte responses against the two-
immunodominant SIV epitopes in terms of their frequencies
among the different tissue compartments. Furthermore, we
FIG. 2. Mamu-A*01/Gag CM9 and/Tat SL8 tetramer binding CD3?/CD8?lymphocytes in multiple lymphoid compartments. The frequency of
Mamu-A*01/Gag CM9 (A) or Mamu-A*01/Tat SL8 (B) tetramer binding cells from multiple lymphoid tissues is shown. The dot plots are gated
on CD3?lymphocytes, but the frequencies are reported as the percentages of CD3?/CD8?lymphocytes. Animal 27099 is shown as an example,
as data from all animals were analyzed in this manner.
VOL. 79, 2005 COMPARTMENTAL SIV IMMUNODOMINANT CD8?T-CELL RESPONSE9231
found no evidence for higher epitope diversity with respect to
the four SIV epitopes in the reproductive tissues and regional
lymph nodes investigated compared to other mucosal sites or
lymphoid tissues (data not shown).
Homing of IFN-?-secreting SIV-specific CD8?T lympho-
cytes to the cervicovaginal mucosa. Because insufficient num-
bers of cells were recovered to directly measure cytotoxicity of
freshly isolated CD8?T lymphocytes from the female repro-
ductive tissues and GALT, we evaluated the effector status and
location of the SIV epitope-specific lymphocytes by ICS for
production of IFN-? and TNF-?. We detected cytokine-secret-
ing cells in all but one of the animals, in larger numbers in the
female reproductive tract than in the LNs and the GALT
(Table 1), as illustrated for animal 27572 (Fig. 5). We speculate
that the effects hormonal changes during the menstrual cycle
have on effector function (8, 35) may be the reason that we
detected tetramer-positive cells but not cytokine-secreting cells
in one animal (24225).
In the accompanying report, we show that systemic infection
is quickly established throughout the LTs and show in this
report that this occurs before a significant virus-specific CD8?
T-lymphocyte response to the major immunodominant SIV
epitopes has developed. Robust CD8?T-lymphocyte re-
sponses against two immunodominant epitopes are detected
only after the peak of viremia and then mainly in the female
reproductive tract but not in the GALT. Moreover, while
strong responses were detected in the three animals infected
for 20 to 21 days, lower frequencies of tetramer-positive cells
were observed in two of the three animals infected for 27 to 28
days, consistent with declining virus-specific CD8?T-lympho-
cyte responses from a peak earlier in infection. The strikingly
different virus-specific CD8?T-lymphocyte response in the
female reproductive tissues and the GALT is consistent with
an increasing body of evidence that suggests that the mucosal
FIG. 3. Frequency of CD3?/CD8?lymphocyte responses against immunodominant SIV epitopes at 20 to 21 and 27 to 28 days postintravaginal
infection. The frequency of tetramer binding CD3?/CD8?lymphocytes isolated from different lymphoid tissues is shown at day 20 to 21 (A) and
day 27 to 28 (B). Mamu-A*01/Tat SL8 tetramer staining is unavailable for samples from animals 30551, 27572, and 24225. From some tissues,
tetramer staining was not available (N/A) because an insufficient number of lymphocytes were isolated or collected on the FACSCalibur for
9232 REYNOLDS ET AL.J. VIROL.
immune system is a distinctly compartmentalized rather than a
single system (15, 20, 34). We found relatively poor virus-
specific responses in the GALT after intravaginal inoculation,
in contrast to a previous study where relatively high frequen-
cies of Gag CM9-specific CD8?T lymphocytes were detected
in the GALT during acute infection after intravenous inocu-
lation (30). In our study, virus-specific CD8?T lymphocytes
were concentrated in the female reproductive tract. The rela-
tively poor CD8?T-lymphocyte response in GALT, compared
to the response in the female reproductive tract, may be due,
at least in part, to the preferential homing of virus-specific
effector cells back to the initial site of infection (20). This
would account for the higher frequency of epitope-specific
responses in the female reproductive tissues and the decreased
frequency of responses at more distal sites. For two reasons, we
think that it is unlikely that the explanation for the low re-
sponse is the loss of CD8?T lymphocytes during isolation
because of the susceptibility of GALT lymphocytes to undergo
apoptosis. (i) The proportion of CD8?T lymphocytes isolated
from the intestinal tissues was high (65 to 90%). (ii) Using
these same isolation procedures, we have detected high fre-
quencies of tetramer-positive cells in GALT in chronically
infected animals and in the acute stages of infection in animals
infected intrarectally (unpublished data), similar to results pre-
viously described by other investigators (26).
The preferential depletion of CD4?T lymphocytes in the
GALT by SIV and HIV may reflect infection of larger numbers
of susceptible CD4?T-lymphocyte targets (2, 4, 31, 33). How-
ever, the poor to nonexistent virus-specific CD8?T lympho-
cytes documented here may be an important contributing fac-
tor in the preferential CD4?T-lymphocyte depletion in
The robust immune response following intravaginal inocu-
lation was somewhat unexpected in light of a recent report that
a recombinant poxvirus strain of modified vaccinia virus An-
kara failed to elicit CD8?T-lymphocyte responses in the fe-
male reproductive tract of mice when the vaccine was admin-
istered intravaginally but did elicit a response after intranasal
inoculation (11). Here, we show that the lower female repro-
ductive tract in primates can be an excellent antigen-specific
FIG. 4. Comparison of IST staining in vagina at 13 days postinfection (dpi) (top panels) to 28 dpi (middle panels) and an example of
tetramer-positive CD8?T cells in lymph tissues (bottom panels). In each set of panels, the left image is Mamu-A*01 Gag181–189CM9 or Tat28–35SL8
tetramer stain (red), the middle image is CD8 antibody stain (green), and the right panel is a merged image of the red and green images. Panels
A to C and D to F are representative images that show staining in vaginal epithelia at 13 dpi (animal 28035), and 28 dpi (27338), respectively. (G
to I) Obturator lymph node from 20 dpi (27357). Images were collected using a 20? objective and are a z-series projection in which Z-scans were
collected at 2-?m intervals into the tissue, with the vaginal epithelium images encompassing 10 ?m of tissue and the lymph tissue image
encompassing 20 ?m of tissue.
VOL. 79, 2005COMPARTMENTAL SIV IMMUNODOMINANT CD8?T-CELL RESPONSE9233
CD8?T-lymphocyte-priming site when exposed to replication-
This compartmental analysis reveals some of the potential
reasons virus-specific CD8?T-lymphocyte responses fail to
fully contain infection in the LTs targeted by SIV and, by
extension, HIV, because of the relatively low frequency of
virus-specific CD8?T lymphocytes in LTs after peak virus
production. However, a vaccine-induced local recall response
might control viral replication before the immune system must
contend with large numbers of infected cells distributed
throughout the LTs. Evidence that a local response might
prevent transmission comes from studies in which antigen-
specific IFN-?-producing CD8?T lymphocytes were detected
in cervical tissue samples of women exposed to seropositive
partners who remain seronegative with no detectable plasma
viral loads (13).
In summary, we show here that SIV replicates to peak levels
unchecked by a CD8?T-lymphocyte response against immu-
nodominant SIV epitopes for at least 1 week and, in the GALT
for an additional 2 weeks following intravaginal transmission.
CD4?T lymphocytes in GALT thus lack protection from in-
fection and the cytopathic effects of SIV replication for an
extended period, which may be one reason that CD4?T-
lymphocyte depletion is greater in GALT than in other lym-
phoid organs. In this experimental model of the natural history
of intravaginal transmission and acute infection, the immune
response is too little, too late, just as it may be in preventing
the establishment of a persistent HIV-1 infection (9). To be
successful, a vaccine would clearly have to induce a much more
rapid and robust recall response, not only in the reproductive
tissues where infection begins but also throughout all LT com-
partments. Nonetheless, the robust response in female repro-
ductive tissue is an encouraging sign that it may be possible to
develop an effective vaccine that limits infection at the portal
FIG. 5. Peptide-specific cytokine production. Cytokine-producing
CD8?lymphocytes are detected at higher frequencies in female re-
productive tissues than in lymph nodes and GALT. Animal 27572 is
used as an example.
TABLE 1. Frequency of peptide-specific IFN-? and TNF-? production by CD8?lymphocytes among the different lymphoid and nonlymphoid tissues, as measured by intracellular
0.01 0.02 ?0
0.04 0.01 ?0
0.15 0.23 0.05 ?0
0.02 0.09 ?0
0.58 0.50 0.47
1.05 1.60 0.18
0.25 0.18 ?0
0.12 0.19 0.59 ?0
0.14 0.02 ?0
0.11 0.21 0.20
0.04 1.01 ?0
0.08 0.86 ?0
0.03 0.08 0.35 ?0
0.33 0.20 ?0
0.08 0.19 ?0
0.03 0.53 ?0
0.45 0.73 0.67
0.40 1.59 ?0
0.24 1.25 ?0
0.20 1.96 ?0
0.10 0.14 1.20 ?0
0.22 1.51 ?0
0.03 0.11 0.51
3.43 1.04 ?0
aPeptide-specific responses are reported after background has been deducted. The column (?) is the frequency of nonspecific cytokine production detected in the medium-only negative control. The symbol ? in the
table represents samples not tested, and ?0 represents samples that were at or below background. IFN-? production was measured in the data shown in the first eight rows, and TNF-? production was measured in the
data shown in the next eight rows.
9234REYNOLDS ET AL. J. VIROL.
ACKNOWLEDGMENTS Download full-text
We thank the Immunology Core Laboratory and Primate Services
Unit of the California National Primate Research Center (CNPRC);
the staff at the Wisconsin Primate Research Center (WPRC); Ding Lu,
Tracy Rourke, Rino Dizon, and Blia Vang for technical assistance; and
Colleen O’Neill and Tim Leonard for help in preparing the manuscript
This work was supported by NIH grants R01 AI48484 to A.H., U51
RR00169 to the CNPRC, R01 AI51239 and R01 AI51596 to C.J.M.,
and P51 RR00167 to the WPRC.
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