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Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
TNF-
, and IL-2 after in vitro exposure to a nonspecific mitogen or
the optimal peptide representing a dominant virus-specific CTL epitope.
However, similarly performed studies assessing these capabilities in
CTL from monkeys infected with pathogenic immunodeficiency virus
isolates demonstrated a significant dysfunction in the ability of the
CTL to produce IL-2 and TNF-. Importantly, CTL from vaccinated
monkeys that effectively controlled the replication of a highly
pathogenic simian-human immunodeficiency virus isolate following
challenge demonstrated a preserved capacity to produce these cytokines.
These experiments suggest that defects in cytokine production may
contribute to CTL dysfunction in chronic HIV or SIV infection.
Moreover, an AIDS vaccine that confers protection against clinical
disease evolution in this experimental model also preserves the
functional capacity of these CTL to produce both IL-2 and
TNF-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now possible to analyze with remarkable precision the phenotypic characteristics and functional capabilities of virus-specific CTL using MHC class I/peptide tetramers (11, 12). Studies employing this technology have detailed a variety of abnormalities in HIV-1-specific CTL that are associated with a loss of cytotoxic function (1, 13). These include abnormalities in the production of mediators of cytotoxicity as well as the expression of cell signaling molecules. The potential contribution of abnormal cytokine production to these defects, however, is still unclear.
Antiviral effects of selected cytokines have been well described. Both
IFN- and TNF- mediate potent antiviral responses (14, 15). In fact, the ability of CTL to secrete these cytokines is
accepted as an indicator of their cytotoxic fitness. Whether
CD8+ CTL in HIV-1-infected individuals have
defects in cytokine secretion remains an unresolved issue.
We have utilized SIV-infected and simian-human immunodeficiency virus
(SHIV)3-infected
rhesus monkeys to investigate the biology of virus-specific CTL in the
clinical setting of AIDS. SIV/SHIV-infected rhesus monkeys that express
the MHC class I allele Mama-A*01 develop a dominant
Gag-specific CTL response focused on the p11C epitope
(16). Fluorochrome-labeled tetrameric
Mamu-A*01/p11C complexes allow the analysis of these
epitope-specific CD8+ CTL by flow cytometric
methods (11, 17). This technique has recently allowed a
precise characterization of the role of CTL in early AIDS virus
clearance, the anatomic compartmentalization of CTL populations, and
the V
gene repertoire utilized by AIDS virus-specific CTL
(18, 19, 20).
The present studies were initiated to assess the functional capacity of
CTL in SIV/SHIV-infected rhesus monkeys. In these experiments, Gag p11C
tetramer-binding CD8+ T lymphocytes from infected
Mamu-A*01+ monkeys were assessed for
their ability to secrete IFN-, TNF-, and IL-2. These studies show
that virus-specific CD8+ T lymphocytes in animals
with progressive disease exhibit a reduced capacity to secrete both
IL-2 and TNF-, and this loss is associated with a high viral load
and low CD4+ T lymphocyte numbers. Interestingly,
these studies also showed that vaccine protection against SHIV-induced
clinical disease in this model was associated with preserved functional
CTL capacity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heparinized blood samples were obtained from rhesus monkeys (Macaca mulatta). All animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (21). Viruses used in this study were the nonpathogenic SHIV-89.6, the J5 strain of SIVmac251 with low pathogenicity, and the highly pathogenic SIVsmE660 and SHIV-89.6P strains. SHIV-89.6 and SHIV-89.6P are chimeric viruses consisting of the SIVmac239 backbone and the HIV-1 89.6 envelope gene, which was cloned from a primary patient R5/X4 dual-tropic HIV-1 isolate (22).
CD4 counts and viral loads
Peripheral blood CD4+ T lymphocyte counts were determined by multiplying the total lymphocyte count by the percentage of CD3+ CD4+ T cells assessed by flow cytometry. Plasmid viral RNA levels were measured by a real-time RT-PCR amplification assay with a detection limit of 400–500 copies/ml, as described (23), using gag primers and probes (24).
Selection of Mamu-A*01+ rhesus monkeys
Rhesus monkeys were screened for the presence of the Mamu-A*01 allele by a PCR-based technique, as previously described (25). EDTA-anticoagulated whole blood from rhesus monkeys was subjected to Ficoll diatrizoate density gradient centrifugation to isolate leukocytes, and the washed cell pellets were resuspended in 200 µl PBS. DNA extraction was then conducted with a QIAmp blood kit (Qiagen, Chatsworth, CA). PCR was performed on 200–500 µg extracted DNA with allele-specific primers in a 50 µl reaction mixture consisting of 60 mM Tris (pH 8.5), 2 mM MgCl2, 15 mM ammonium sulfate, 2 mM deoxynucleoside triphosphates (0.5 mM each), and 5 µl Taq polymerase. Primers A*01/F (5'-GAC AGC GAC GCC GCG AGC CAA-3') and A*01/R (5'-GCT GCA GCG TCT CCT TCC CC-3') were used at final concentrations of 800 nM each. Two additional primers specific for a conserved MHC class II sequence (based on the rhesus monkey homologue of HLA-DRB3) were included in the reaction as an internal positive control. Primers 5' MDRB (5'-GCC TCG AGT GTC CCC CCA GCA CGT TTC-3') and 3' MDRB (5'-GCA AGC TTT CAC CTC GCC GCT G-3') were used at final concentrations of 680 nM each. PCR was conducted with a GeneAmp System 9600 thermocycler (PerkinElmer, Norwalk, CT). Samples were denatured at 96°C for 2 min, followed by 5 cycles of 25 s at 96°C and 60 s at 72°C; 21 cycles of 25 s at 96°C, 50 s at 67°C, and 45 s at 72°C; and 4 cycles of 25 s at 96°C, 60 s at 55°C, and 80 s at 72°C. The PCR products were analyzed by 1% agarose gel electrophoresis. Ten microliters of each PCR reaction mixture were loaded per lane.
Potential Mamu-A*01-positive animals were identified by the presence of two bands, a 685- and a 260-bp amplified product. DNA sequence analysis was then performed on all potential positive samples to confirm nucleotide sequence identity with the published Mamu-A*01 prototype sequence (16). Before being sequenced, the amplified DNA was treated with 1 U per reaction of shrimp alkaline phosphatase and 10 U exonuclease I for 15 min at 37°C, followed by 15 min at 80°C. The sequencing templates were then purified with a QIAquick PCR purification kit (Qiagen). For each template, 70 ng DNA was used for DNA sequencing together with 5 pmol primer. Four PCR primers were used for sequencing: A*01/F and A*01/R, whose sequences are shown above, and B/1+ (5'-CTG CGC GGC TAC TAC AAC CA-3') and G/1+ (5'-ATG TAA TCC TTG CCG TCG TA-3'). Sequencing was conducted at a central core sequencing facility on an ABI-373 stretch DNA-sequencing machine, using ABI AmpliTaq FS dye terminator chemistry (PerkinElmer). All animals used in this study were genotypically Mamu-A*01 positive based on the above screening, and were also Mamu-A*01 positive as determined in a functional CTL assay.
Plasmid DNA vaccination protocol
The detailed vaccination protocol has been previously described (26, 27). Briefly, double-CsCl-banded maxipreparations of HIV-1 89.6P env (KB9) and SIVmac239 gag plasmid DNA were injected into both quadriceps muscles of rhesus monkeys. Animals treated with IL-2/Ig protein were inoculated twice daily with 0.5 mg/day for 14 days after DNA vaccine administration. Other animals received 5 mg IL-2/Ig plasmid on day 2 after DNA vaccination. The vaccination regimen consisted of inoculation at week 0, followed by boost immunizations at wk 4, 8, and 40. IL-2 treatments were included only at wk 0 and 4. Animals were challenged at wk 46 with 100 monkey infectious dose 50% (MID50) SHIV-89.6P by the i.v. route.
Mamu-A*01/peptide tetramer complex formation and staining of PBMCs
Mamu-A*01/p11C tetramer complexes were prepared as previously
described (11, 17). PE-labeled streptavidin (Prozyme, San
Leondro, CA) was mixed stepwise with biotinylated Mamu-A*01/p11C
peptide complexes at a molar ratio of 1:4 to produce the tetrameric
complexes. All Abs used in this study were directly coupled to FITC,
PE-Texas Red (ECD), or allophycocyanin. The following mAbs were
used: anti-CD3 FITC (SP34; BD PharMingen, San Diego, CA),
anti-CD8 ECD (7PT; Beckman Coulter, Fullerton, CA),
anti-IFN- allophycocyanin (B27; BD PharMingen), anti-TNF-
allophycocyanin (MAb11; BD PharMingen), and anti-IL-2
allophycocyanin (MQ1–17H12; BD PharMingen).
PBMC stimulation and intracellular cytokine staining
Unfractionated fresh PBMCs were cultured at 37°C in a 5% CO2 environment for 6 h in the presence of RPMI medium alone (unstimulated) or RPMI medium containing 5 µg/ml p11C (CTPYDINQM) minimal peptide (peptide stimulated) or 100 ng/ml PMA + 1 µg/ml ionomycin (PMA + Iono stimulated). All cultures contained brefeldin A (10 µg/ml, GolgiPlug; BD PharMingen) to disrupt Golgi apparatus transport, thereby causing the accumulation of intracellular cytokines, as well as 2 µg/ml anti-CD28 and anti-CD49d. The cultured cells were stained with the Mamu-A*01/p11C tetramer and mAbs specific for cell surface molecules before RBC lysis and fixation (Immunoprep reagent system and Q-prep workstation; Beckman Coulter). The PBMCs were washed once with PBS/2% FCS, then permeabilized with Cell Fix/Perm solution (BD PharMingen), according to the permeabilization protocol. Cells were washed twice with 2.5 vol 1x Perm/Wash buffer (BD PharMingen) and then stained with 1 µg anti-cytokine mAb/106 cells. Anti-cytokine mAbs were titered for optimal staining in preliminary experiments. Cells were washed twice with 2 ml 1x Perm/Wash buffer and once with PBS, and then fixed in 1.5% formaldehyde/PBS. Samples were analyzed on the FACSCalibur instrument using CellQuest software. Data analysis was performed using CellQuest software and presented in Microsoft PowerPoint 98 (Microsoft, Redmond, WA).
Statistical analyses
The mean cytokine expression percentages of each group were compared with two-sided Wilcoxon rank sum tests with Bonferroni adjustments of p values for the two major comparisons. A p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs specific for human cytokines were assessed to determine
whether they were suitable reagents for intracellular staining
procedures to detect rhesus monkey cytokines. Selected mAbs specific
for IFN-, TNF-, and IL-2 stained permeabilized human and monkey
PBMCs with equal intensity (Fig. 1
), and
were therefore used in all subsequent experiments.
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, TNF-, and IL-2,
and evaluated by flow cytometric analysis, gating on p11C
tetramer+CD3+CD8+
lymphocytes. These cells were cultured with a Golgi apparatus inhibitor
so as to prevent cytokine secretion and allow the accumulation of
cytokine protein in their disrupted Golgi network. Cells that received
no in vitro stimulus demonstrated little accumulation of cytokines. The
positive control PMA and ionomycin stimulation induced high levels of
cytokine accumulation in these gated p11C
tetramer+CD3+CD8+
T lymphocytes (Fig. 2). In cells
stimulated with the SIV Gag p11C peptide, significant cytokine
accumulation was seen in the Golgi network of the gated CTL. These
observations suggested that this approach could be utilized to assess
the potential for cytokine production by rhesus monkey CTL.
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We first sought to assess the potential for cytokine production by
CTL in a group of clinically healthy
Mamu-A*01+ monkeys infected with the
nonpathogenic SHIV-89.6 or an SIVmac251(J5) isolate with a relatively
attenuated pathogenic potential. These monkeys had been infected for
more than 2 years, had maintained low viral loads and relatively
normal CD4+ T cell counts, and showed no clinical
signs of disease progression (Table I).
PBMCs from all of these animals contained detectable populations of
p11C tetramer+CD8+ T
cells. Upon stimulation, these
tetramer+CD8+ T cells
expressed high levels of IFN- and TNF-, and moderate levels of
IL-2 (Fig. 3A). Specific Gag
epitope peptide stimulation induced IFN- expression in 80% (median;
range 66–96%), TNF- expression in 88% (median; range 78–98%),
and IL-2 expression in 42% (median; range 21–47%) of the p11C
tetramer+CD8+ T cells. The
maximal potential cytokine secretion of these epitope-specific cells
was determined by PMA and ionomycin stimulation. Most of these
lymphocytes were able to produce IFN- (median 98%; range 95–100%)
and TNF- (median 90%; range 59–98%), while a lower percentage
(median 54%; range 31–86%) was capable of IL-2 production.
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We then analyzed the cytokine expression profile of CTL in a group
of Mamu-A*01+ monkeys infected with
pathogenic SIV or SHIV on day 150 postchallenge. These animals had high
viremia and clear signs of progressive disease (Table I
). A total of
62% (median; range 33–79%) of the p11C
tetramer+CD8+ T cells
expressed IFN-, and 66% (median; range 21–69%) expressed TNF-,
while only 10% (median; range 3–14%) expressed IL-2 following
stimulation by Gag epitope peptide. Similarly, 92% (median; range
75–97%) of these same cells expressed IFN- and 67% (median; range
47–85%) expressed TNF-, and 10% (median; range 4–24%) expressed
IL-2 when stimulated by the polyclonal activators PMA and ionomycin
(Fig. 3B). A statistical analysis using the Wilcoxon rank
sum test with Bonferroni adjustments of p values
demonstrated that the mean specific peptide-induced IL-2 and TNF-
production of this group of animals infected with pathogenic viral
isolates differed significantly from the group infected with
nonpathogenic/pathogenically attenuated isolates
(p = 0.03) (Fig. 4). Therefore, the capacity of
CD8+ CTL to produce IL-2 following both specific
and nonspecific stimulation was substantially diminished in the
peripheral blood of these chronically infected monkeys.
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Finally, we assessed cytokine production by CTL in a group of
monkeys that were vaccinated and then challenged with the highly
pathogenic SHIV-89.6P virus on day 150 postchallenge. These animals had
been vaccinated with plasmid DNA constructs expressing HIV-1 89.6P Env
(KB9) and SIVmac239 Gag augmented with either IL-2/Ig protein or
an IL-2/Ig plasmid. Two further monkeys from this vaccination trial,
animals that received a sham vaccine (KPB) or the DNA vaccines
alone (811), are included in the cohort of monkeys evaluated as
animals with progressive disease (Table I, Fig. 3B). Upon
stimulation with Gag epitope peptide, 66% (median; range 51–76%) of
the p11C
tetramer+CD8+ T cells
from this group of vaccinated and challenged animals expressed IFN-,
80% (median; range 78–90%) expressed TNF-, and 27% (median;
range 18–63%) expressed IL-2. The potent polyclonal stimulation
resulting from PMA and ionomycin treatment induced 92% (median; range
86–96%) of the p11C
tetramer+CD8+ T cells
to produce IFN-, 91% (median; range 79–99%) to produce TNF-,
and 25% (median; range 13–46%) to produce IL-2 (Fig. 3C).
The statistical comparison of this vaccinated group to the untreated
animals that similarly received a pathogenic virus infection
demonstrated a highly significant difference (p
= 0.008) (Fig. 4). Thus, the cytokine expression profiles of CTL from
these vaccinated and challenged animals were similar to those of the
clinically healthy animals infected with nonpathogenic or
pathogenically attenuated viruses. These data demonstrate that CTL of
monkeys that have been effectively vaccinated before challenge with a
highly pathogenic SHIV maintain their capacity to produce IL-2 and
TNF- in response to either Gag epitope-specific or nonspecific
mitogen stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(32).
In fact, the ability of Ag-specific CD8+ CTL to
produce IFN- following Ag stimulation has become a surrogate
indicator of the functional status of these cells.
In this study, we demonstrate a statistically significant reduction in
the ability of virus-specific CD8+ CTL to produce
IL-2 and TNF- in monkeys with progressive clinical disease (Fig. 4).
A statistically significant decreased capacity of these cells to
produce IFN- was not seen. These data suggest that rather than using
IFN- production as an indicator of virus-specific
CD8+ CTL fitness, it may be more useful to
determine the capacity of these cells to produce IL-2 and/or
TNF-.
Other studies have also demonstrated a loss of the ability of virus-specific CD8+ CTL to produce IL-2 in this clinical setting (10). It has been suggested that this dysregulation in IL-2 production is associated with a lack of CD4+ T cell help, and that expansion of virus-specific CD8+ T lymphocytes in the absence of IL-2 can lead to the establishment of anergy in these cells (10, 33). While we saw low IL-2 production by CD8+ CTL in the cohort of animals that had high viral loads and low CD4+ T lymphocyte counts, these data do not differentiate between these potential mechanisms. Thus, a loss of the capacity of CTL to produce IL-2 may reflect a loss of the CD4+ T cell help needed to expand virus-specific CD8+ CTL or a specific defect in the functional repertoire of the virus-specific CD8+ CTL population.
Importantly, the virus-specific CD8+ CTL from all
the animals that were clinically healthy following infection with
pathogenically attenuated or nonpathogenic SIV and SHIV isolates
produced normal levels of IL-2 and TNF-. In addition, the monkeys
that were vaccinated before infection also showed normal levels of IL-2
and TNF- production by their virus-specific
CD8+ CTL. These animals were immunized with DNA
vaccines augmented by IL-2/Ig protein or IL-2/Ig plasmid before
challenge and exhibited significant control of viremia and prevention
of clinical disease progression (Table II) (27). These vaccinated
animals had CTL that exhibited levels of IL-2 and TNF production that
were significantly higher than the levels observed in the CTL of
typical progressors (p = 0.008), two of which
were similarly infected with SHIV-89.6P. In fact, the levels of
cytokine production in the vaccinated animals were comparable with the
levels observed in the clinically healthy animals infected with
nonpathogenic/pathogenically attenuated virus isolates. Thus, effective
vaccination of rhesus monkeys before a pathogenic viral challenge
preserved their capacity to produce cytokines in response to specific
Ags and established a phenotype comparable with the animals infected
with pathogenically attenuated and nonpathogenic viruses.
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production may not be as sensitive as
the determination of IL-2 and TNF- production as an indicator of
virus-specific CD8+ T cell function. The
development of in vitro assays such as an ELISPOT for quantification of
the IL-2 and TNF- responses in virus-specific T cell populations may
prove useful in addition to assays for IFN- production to help
determine the functional capacity of HIV-1-specific CTL.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Norman L. Letvin, Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Research East Room 113, 330 Brookline Avenue, Boston, MA 02215. E-mail address: nletvin{at}caregroup.harvard.edu
3 Abbreviation used in this paper: SHIV, simian-human immunodeficiency virus.
Received for publication July 9, 2001. Accepted for publication October 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon. J. Virol. 74:10249.
and CD28, key signaling molecules for T-cell activation. J. Virol. 74:7320.-activated monocytes impair infectivity of HIV particles by an oxygen metabolite-dependent reaction. Immunology 78:171.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), SIV Gag p11C
peptide (
), or PMA and ionomycin (
). IFN-