Functional Impairment of Simian Immunodeficiency Virus-Specific CD8+ T Cells during the Chronic Phase of Infection

Abstract

In an attempt to determine why high frequencies of circulating virus-specific CD8+ T cells are unable to control human immunodeficiency virus and simian immunodeficiency virus (SIV) replication, we assessed
the functional nature of SIV-specific CD8+ lymphocytes. After vaccination and early after infection, nearly all tetramer-staining CD8+ cells produced gamma interferon in response to their specific stimulus. However, by 4 months postinfection with pathogenic
SIVmac239, signs of functional impairment in the CD8+ T-cell compartment were detected which might prevent these T cells from efficiently controlling the infection during the
chronic phase.

Full text

JOURNAL OF VIROLOGY,
0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.5.2458–2461.2001
Mar. 2001, p. 2458–2461 Vol. 75, No. 5
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Functional Impairment of Simian Immunodeficiency Virus-
Specific CD8
⫹
T Cells during the Chronic Phase of Infection
THORSTEN U. VOGEL,
1
TODD M. ALLEN,
1
JOHN D. ALTMAN,
2
AND DAVID I. WATKINS
1,3
*
Wisconsin Regional Primate Research Center
1
and Department of Pathology and Laboratory Medicine,
3
University of
Wisconsin, Madison, Wisconsin 53715, and Emory Vaccine Center, Emory University School of Medicine,
Atlanta, Georgia 30322
2
Received 2 October 2000/Accepted 7 December 2000
In an attempt to determine why high frequencies of circulating virus-specific CD8
ⴙ
T cells are unable to
control human immunodeficiency virus and simian immunodeficiency virus (SIV) replication, we assessed the
functional nature of SIV-specific CD8
ⴙ
lymphocytes. After vaccination and early after infection, nearly all
tetramer-staining CD8
ⴙ
cells produced gamma interferon in response to their specific stimulus. However, by
4 months postinfection with pathogenic SIVmac239, signs of functional impairment in the CD8
ⴙ
T-cell
compartment were detected which might prevent these T cells from efficiently controlling the infection during
the chronic phase.
It is still unclear why the immune system is not able to clear
an infection with immunodeficiency viruses. These lentiviruses
appear to have devised multiple strategies to evade the im-
mune response (reviewed in reference 26), including major
histocompatibility complex class I downregulation (10) and
escape from cytotoxic T lymphocyte (CTL) responses (1, 7, 9,
13, 17, 27). However, the maintenance of high frequencies of
virus-specific cells against certain viral epitopes in human im-
munodeficiency virus (HIV)-infected humans (4, 23, 30) and
simian immunodeficiency virus (SIV)-infected monkeys (19,
20) indicates that these epitopes are still being recognized and
have not mutated. While the invention of tetramers (4) made
it possible to detect these high frequencies of virus-specific
cells in HIV and SIV infection, tetramer staining simply iden-
tifies antigen-specific lymphocytes but does not provide infor-
mation about the functional nature of these virus-specific cells.
Functionally impaired CD8
⫹
T-cell responses have been pre-
viously described by Zajac et al. in chronic lymphocytic cho-
riomeningitis virus (LCMV) infection (31) and by Lee et al. in
a tumor system (22), where CTL were unable to directly lyse
their specific target cells and produce cytokines in response to
mitogens. We therefore were interested in investigating
whether similar functional defects could account for the inabil-
ity of CD8
⫹
T cells to control HIV and SIV infections. To
address this question we combined tetramer-staining technol-
ogy (4) and the intracellular cytokine assay (18, 25) to deter-
mine whether SIV-specific CD8
⫹
T cells manifest any func-
tional defects.
The majority of tetramer-positive cells produce IFN-␥after
peptide-specific stimulation in vaccinated animals and early
after infection with pathogenic SIVmac239. Using an epitope-
based DNA prime-modified vaccinia virus Ankara boost vac-
cine, we induced Mamu-A*01-restricted, p11C,C-M (CM9)-
specific CTL (2) in three Mamu-A*01-positive rhesus
macaques (95058, 95045, and 96031), as previously described
(3). We followed the levels of Mamu-A*01/CM9-specific cells
in these animals before and after intrarectal infection with
SIVmac239 (molecular clone) by tetramer staining and inves-
tigated their ability to produce gamma interferon (IFN-␥) after
antigen-specific stimulation using the intracellular cytokine as-
say. Fresh or thawed peripheral blood mononuclear cells (PB-
MCs) were stimulated for 6.5 h with mitogen (50 ng of phorbol
myristate acetate/ml and 1 ␮g of ionomycin/ml) or with 5 ␮M
CM9 peptide in the presence of B-lymphoblastoid cell line
cells (B-LCL) as antigen-presenting cells. Brefeldin A (10 ␮g/
ml) was present for the last5htoinhibit the secretion of any
produced cytokines. The cells were then surface stained with
anti-CD8 antibodies (conjugated to peridinin chlorophyll pro-
tein; Becton-Dickinson) alone or together with the Mamu-
A*01/CM9 tetramer (labeled with phycoerythrin) for 40 min at
room temperature. The cells were then washed with flow buffer
(2% fetal calf serum in phosphate-buffered saline), fixed with
paraformaldehyde (2% in phosphate-buffered saline) over-
night, permeabilized with 0.1% saponin, and stained intracel-
lularly with anti-IFN-␥antibodies (labeled with fluoroscein
isothiocyanate; Pharmingen), as described previously (3). As
the T-cell receptor is downregulated after antigen-specific
stimulation (29), the tetramer staining of CM9-specific cells
nearly completely disappeared following CM9-specific stimu-
lation. Therefore, we were not able to express the percentage
of tetramer-positive cells able to produce IFN-␥. However, we
observed that in immunized animals and early after infection,
the percentage of CD8
⫹
cells expressing IFN-␥after peptide-
specific stimulation correlated with the levels of tetramer stain-
ing in unstimulated samples (Fig. 1; see also reference 3). All
tetramer-positive lymphocytes produced IFN-␥at weeks ⫺3, 0,
and 2 postchallenge (the ratio of IFN-␥-producing cells to
tetramer-positive cells was approximately 1). We also observed
this correlation between the intracellular cytokine assay and
tetramer staining in other immunized animals and for another
Mamu-A*01-restricted epitope (data not shown). Between
weeks 6 to 8 postchallenge, some tetramer-positive cells from
* Corresponding author. Mailing address: Wisconsin Regional Pri-
mate Research Center, 1220 Capitol Ct., Madison, WI 53715. Phone:
(608) 265-3380. Fax: (608) 265-8084. E-mail: watkins@primate.wisc
.edu.
2458

all three animals (95058, 95045, and 96031) were unable to
produce IFN-␥(ratios below 1). However, by week 16 the
ratios came back up to values around or higher than 1. As this
drop was only temporary, it is not clear if this represents a
significant change in phenotype and could indicate a functional
impairment of tetramer-positive cells shortly after the primary
peak of viremia reached its high point in week 3 (Allen et al.,
unpublished observations). However, the two naive control
animals (95114 and 95115), which were infected with the same
virus at the same time as the immunized animals (Allen et al.,
unpublished), also evidenced a reduced ratio during the time
of primary viremia, which peaked at week 4 postchallenge.
This may indicate an impaired ability of some tetramer-posi-
tive cells to produce IFN-␥shortly after peak viral replication
was resolved in these animals. Nevertheless, the ratios re-
turned to values of approximately 1 in week 16 in these two
control animals (Fig. 1). These results indicate that the major-
ity of tetramer-positive cells produce IFN-␥in response to
stimulation with their cognate peptide up to 4 months after
infection with pathogenic SIV.
Discrepancy between tetramer staining and intracellular
cytokine production after 4 months of infection with patho-
genic SIVmac239. Unexpectedly, the number of tetramer-pos-
itive cells that produced IFN-␥in response to peptide-specific
stimulation dropped dramatically after 4 months of infection
(Fig. 1). The ratio of IFN-␥-positive cells to tetramer-positive
cells dropped below 0.66 in all animals and remained relatively
stable at these lower values thereafter. There was no obvious
difference between previously immunized and naive control
animals. The possibility that the reduced cytokine production
is caused by a destruction of antigen-presenting cells was ex-
cluded, because B-LCL were added as antigen-presenting cells
in the intracellular cytokine assay. It is, therefore, likely that
the virus-specific cells during chronic SIV infection have de-
fects in cytokine production in response to stimulation by their
cognate peptide. Interestingly, we were able to demonstrate
that most of the tetramer-positive cells in our SIV-infected
macaques were still able to produce IFN-␥after mitogen stim-
ulation at week 24 (data not shown) and at week 36 postchal-
lenge (Fig. 2). This contrasts with the “silent” phenotype de-
scribed by Zajac et al. in chronic LCMV infection (31) and by
Lee et al. in a tumor system (22). Therefore, the defect of the
virus-specific cells in our monkeys is not as prominent, at least
at this early stage of infection with SIV, as the defect observed
by Zajac and colleagues in LCMV-infected mice (31). It is
interesting that Donahoe and colleagues found a good corre-
lation of intracellular cytokine production after peptide-spe-
cific stimulation, in this case tumor necrosis factor alpha, and
Mamu-A*01/CM9-tetramer staining in monkeys infected with
an attenuated, nonpathogenic SIV, SIVmac239delta nef (11).
It is possible that tetramer-positive cells in animals infected
with an apathogenic SIV might not demonstrate a reduced
ability of cytokine production as found in our animals, but this
needs to be investigated further.
Similarities to functional defects of LCMV- and HIV-spe-
cific immune responses. Zajac and colleagues also showed that
the silent phenotype of virus-specific CTL was especially prev-
alent when there was inadequate CD4 help. Infection with SIV
or HIV does impair CD4 help (5, 12, 21, 24), and it may
therefore not only result in a loss of virus-specific CD8 T-cell
response over time (8, 15) but also result in a functional im-
pairment of the virus-specific CTL response. Although the
absolute numbers of CD4-positive cells per microliter of blood
in our SIV-infected monkeys were continuously decreasing
over time (data not shown), there was no obvious correlation
between the drop in IFN-␥production in tetramer-positive
cells with CD4 counts. A recent study by Gea-Banacloche and
colleagues described an inability of 30 to 50% of tetramer-
FIG. 1. Functional nature of tetramer-positive, CD8
⫹
T cells. PBMCs were stimulated with the CM9 peptide for 6.5 h, with the last5hinthe
presence of BFA. The percentage of CD8
⫹
cells producing IFN-␥(IFNg), as detected in the cytokine assay, was divided by the percentage of
CD8-positive cells staining with the Mamu-A*01/CM9-tetramer before stimulation, to obtain the ratios plotted. Only values of ⬎0.1% (above
background) of CD8
⫹
cells were considered positive for both the tetramer staining and the intracellular cytokine assay (see Fig. 2 legend). Ratios
below 1 indicate that some tetramer-positive cells are unable to produce IFN-␥in response to specific peptide stimulation. Weeks ⫺3, 6, 24, 28,
40, 44, and 48 were tested using thawed PBMCs, and samples from week 6, 28, 44, and 48 were all tested in one assay.
VOL. 75, 2001 NOTES 2459

positive cells to produce IFN-␥in response to stimulation with
recombinant vaccinia virus-infected B-LCL, which expressed
HIV genes, in two HIV-infected patients (14), which is very
similar to the defect of SIV-specific CD8
⫹
T cells described
here. In addition, Goepfert and colleagues have described a
10-fold difference between the number of peptide-specific cells
as measured by tetramer staining or ELISPOT assay in HIV-
infected patients (16). Therefore, as we have demonstrated
here, SIV infection seems to cause a functional impairment of
the specific immune response that is very similar to that which
has been described for HIV infection (6, 14, 16, 28). The
SIV/rhesus monkey model is well suited for investigating the
mechanism behind the impairment of the virus-specific im-
mune response. Finally, our finding that not all antigen-specific
CD8
⫹
lymphocytes effectively produce IFN-␥during the
chronic phase of infection has implications for the measure-
ment of antigen-specific CD8
⫹
lymphocytes during chronic
SIV infection. Intracellular staining for IFN-␥might result in
the underestimation of antigen-specific CD8
⫹
lymphocytes in
the chronic phase of SIV infection if this is the sole assay
employed. Multiple assays, therefore, need to be employed
when assessing antiviral immune responses in chronically in-
fected macaques and humans.
This work was supported by grants AI42512, AI41913, and
RR00167. D.I.W. is a recipient of an Elizabeth Glaser Scientist award.
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FIG. 2. IFN-␥production in response to mitogen or peptide-specific stimulation at week 36 postchallenge. Tetramer-positive cells are still able
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