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Laboratories of
*
Immunoregulation and
Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Science Applications International Corp., Frederick Cancer Research and Development Center, Frederick, MD 21702;
§
Emory University Vaccine Center at Yerkes, Atlanta, GA 30322; and
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Servicio de Medicina Interna 1, Clinica Puerta de Hierro, Universidad Autonoma de Madrid, and Servicio de Microbiologia, Hospital General Gregorio Maranon, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to HIV-vaccinia recombinant-infected
autologous B cells. Very high frequencies (0.8–18.0%) of circulating
CD8+ T cells were found to be HIV specific. High
frequencies of HIV-specific CD8+ T cells were not limited
to long-tern nonprogressors with restriction of plasma virus. No
correlation was found between the frequency of HIV-specific
CD8+ T cells and levels of plasma viremia. In each case,
the vast majority of cells (up to 17.2%) responded to
gag-pol. Repertoire analysis showed these large numbers
of Ag-specific cells were scattered throughout the repertoire and in
the majority of cases not contained within large monoclonal expansions.
These data demonstrate that high numbers of HIV-specific
CD8+ T cells exist even in patients with high-level viremia
and progressive disease. Further, they suggest that other qualitative
parameters of the CD8+ T cell response may differentiate
some patients with very low levels of plasma virus and nonprogressive
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of techniques have recently become available that allow the measurement of the magnitude of such responses by determining the number of Ag-specific CD8+ T cells. MHC tetramers permit the determination at the single-cell level of CD8+ T cells specific for a given peptide (13). These reagents have dramatically revised the estimates of the magnitude of the Ag-specific CD8+ T cell response during acute infections of mice and humans as much as 10- to 100-fold above that previously found by traditional limiting dilution analysis. It has been determined that 40–70% of the CD8+ T cells during an acute infection of experimental animals or humans may be Ag specific (14, 15, 16, 17). The total numbers of Ag-specific CD8+ T cells during chronic infections of humans have not yet been well characterized. MHC tetramer analysis has recently permitted the quantification of HIV peptide-specific CD8+ T cells in the peripheral blood in some patients with progressive HIV disease (18). However, tetramer analysis alone allows the determination of the number of cells specific for a given peptide. It is likely the true number of Ag-specific CD8+ T cells is much larger if one were to examine the response to all HIV gene products in the context of each of the patient’s MHC class I alleles. Further, although MHC tetramer complexes are powerful reagents for examining the frequency of Ag-specific cells, such analyses provide no direct information on their functional state.
In the present study, we use a combination of assays to examine the
frequency and function of Ag-specific cells in a detailed analysis of
21 HIV-infected patients. Several of these patients are part of a
unique cohort characterized by infection >13 years, normal
CD4+ T cell counts, HIV RNA below 50 copies/ml of
plasma, and vigorous HIV-specific proliferative and direct CTL
responses. These patients likely make up <0.8% of HIV-infected
individuals (19, 20, 21, 22). For comparison, we also include
patients that fit the more commonly used clinical definition of
nonprogressor and patients with progressive disease. We analyze the
CD8+ T cell response by standard cytotoxic T cell
assays, MHC tetramer analysis, and TCR repertoire analysis. In
addition, we have combined techniques of flow cytometric detection of
intracellular IFN- production with Ag presentation by HIV-vaccinia
recombinant-infected autologous B cells. In this manner, we are better
able to determine the global response to multiple HIV Ags and the
functional state of Ag-specific cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients 1, 4–8, 19–21, 25, 27, and 29 have not received
antiretrovirals during or before the study period. Patient 3 previously
received IFN-
/AZT (1/90 to 12/95) or IFN-/AZT/DDI (1/96 to 12/96)
as part of a National Institute of Allergy and Infectious Diseases
protocol. This patient has remained off of antiretrovirals since that
time. Patients 15 and 14 have not received antiretrovirals in the past
6 mo. HIV infection in study participants was documented by HIV-1/2
enzyme immunoassay. All subjects signed informed consent approved by
the National Institute of Allergy and Infectious Disease
investigational review board. Patients 1–6 were recently described in
a separate report (23). Patients 3–8, 25, and 101–105
have also been reported separately (24). The patient
numbers remain the same across studies to permit cross-reference. HLA
class I and II typing was performed by hybridization with
sequence-specific oligonucleotide probes following amplification of the
corresponding genes using PCR as described elsewhere (25).
CCR5 deletion mutations were detected as previously described
(26).
Cytotoxic T cell assays
PBL were obtained by sodium diatrizoate density centrifugation (Organon-Teknika, Durham, NC) of apheresis donor packs. PBMC were cryopreserved in RPMI 1640 media with 10% FBS and 7.5% DMSO at -140°C. Standard 51Cr release assays were performed as previously described (27). Autologous EBV-transformed B cells were infected for 16 h at 37°C with vaccinia recombinant viruses vVK1 (containing the HIV-1HXB2 gag-pol gene), vP1287 (HIV-1IIIB gag), vP1289 (HIV-1IIIB p24), vP1290 (HIVIIIB p17), vP1288 (HIVIIIB pol), vPE16 (HIV-1BH10 env), vTFnef (HIV-1pNL432 nef), or the negative control virus vSC8 (Escherichia coli ß-galactosidase (ß-gal)2). Vaccinia recombinants were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The vP1287 (HIV-1IIIB gag), vP1289 (HIV-1IIIB p24), vP1290 (HIVIIIB p17), and vP1288 (HIVIIIB pol) viruses were contributed to the National Institutes of Health AIDS Research and Reference Reagent Program by Virogenetics (Troy, NY). The vTFnef virus was contributed by MedImmune (Gaithersburg, MD), and vPE16, vVK1, and VSC8 viruses were contributed by Dr. Bernard Moss (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Target cells were 51Cr labeled, washed, and plated at 1 x 104 cells per well into 96-well round-bottom tissue culture plates. Fresh or cryopreserved PBMC were used as effectors. Cryopreserved PBMC were cultured overnight at 37°C before use as effectors. Preliminary experiments have yielded similar results with fresh or cryopreserved PBMC. Effector PBMC were added to each well at 100–12.5:1. All CTL assays were performed in triplicate. The percent specific cytotoxicity was calculated as follows: % specific cytotoxicity = [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100. The lysis of VSC8-infected targets was 12% for patient 5 and <5% in all other patients in each experiment. Spontaneous release was <20% in each experiment. The SE of individual triplicates was 1–20% of mean specific lysis. In preliminary experiments, significant lysis (>10%) was not observed in MHC class I-mismatched or CD8+ T cell-depleted cultures. All experiments were repeated at least once with similar results.
Flow cytometry
Four-color flow cytometry was performed according to standard
protocols (28). Surface or intracellular staining was
performed using the following Abs: FITC-conjugated anti-IFN- and
anti-CD8 (PharMingen, Cupertino, CA), anti-BV8 and
anti-BV5.1 (T Cell Diagnostics, Woburn MA); PE-conjugated
anti-IFN- (PharMingen), and anti-CD69 (Becton Dickinson, San
Jose, CA) and CyChrome-conjugated anti-CD8 and anti-CD3
(Coulter, Miami, FL), phycoerythrin-Texas Red-conjugated anti-CD8 and
anti-CD3 (Coulter), peridinin chlorophyl protein-conjugated
anti-CD3 and anti-CD8 (Becton Dickinson). Cells were analyzed
within 24 h using either an EPICS XL (Beckman Coulter, Fullerton,
CA) or a FACScalibur (Becton Dickinson) flow cytometer. Surface
staining with anti-BV5.1, anti-BV8, or tetramers was performed
following stimulation but before fixation. Then, 0.5 µl of
APC-conjugated HLA-A*0201 tetrameric complex was used to stain 2
x 106 PBMC in a 50-µl volume at 4°C for 30
min. MHC class I A*0201 complexed with either of the conserved
gag-SLYNTVATL or pol-ILKEPVHGV peptides have been
previously described to stain class I-restricted cytotoxic T cells
specific for these peptides (18).
Intracellular cytokine detection
Target cells were prepared as described above for the direct CTL assay and used as the stimulus for intracellular cytokine detection. Intracellular cytokine detection was performed as previously described (29). Briefly, 4 x 106 PBMC were incubated with 400,000 uninfected, vac-ß-gal, or vac-HIV-recombinant-infected autologous EBV-transformed B cells in a final volume of 2 ml of RPMI 1640 containing 10% FBS in 10-ml culture tubes (Sarstedt, Newton, NC). At 2 h of incubation, brefeldin- A (Sigma, St. Louis, MO) was added to the medium at a final concentration of 10 µg/ml to inhibit cytokine secretion. At 6 h of incubation, the cells were washed twice and fixed in 4% paraformaldehyde (Sigma) and permeabilized or frozen for future use.
Fixed cells were permeabilized and blocked in a solution of PBS with 0.2% saponin (25% sapogenin in content; Sigma), 1 mM CaCl2, 1 mM MgSO4, 0.05% (w/v) NaN3, 1% BSA, pH 7.4, with 5% nonfat dry milk overnight at 4°C. Cells were then aliquoted at 1 x 106 per tube and washed once in a solution of PBS/saponin. The pellet was resuspended in PBS/saponin/milk containing Abs for staining and incubated for 30 min at 4°C in the dark. Samples were then washed twice in PBS/saponin and resuspended in 300 µl PBS/BSA 0.1%. Gating was performed on CD3+CD8+ lymphocytes, and 15,000–200,000 events (100,000–700,000 total cells) were collected. Data were analyzed using either CellQuest (Becton Dickinson) or FlowJo software (TreeStar, Cupertino, CA). Color compensation settings were made with each round of staining using patient cells labeled singly with anti-CD3 labeled with specific fluorochromes.
Repertoire analysis
CD4+ T cells were isolated by depleting monocytes and CD8+ T cells using anti-CD14- and anti-CD8-coated magnetic beads (Dynal, Lake Success, NY). CD4+ T cells were then positively selected with CD4-specific beads. CD8+ T cells were positively selected directly from PBMC with CD8-specific beads. CD4+ or CD8+ T cell purity was documented by flow cytometry to be 95–99%. Total RNA from 107 CD4+ or CD8+ T cells was isolated using Trizol LS (Life Technologies, Grand Island, NY) and precipitated with isopropanol in the presence of Microcarrier (Molecular Research Center, Cincinnati, OH). Reverse transcription was performed using Superscript II (Life Technologies) and oligo(dT) according to manufacturer protocols. Then, 5 µg of total RNA was used for analysis of TCRBV subfamily size patterns, and 1.5 µg of total RNA was used for semiquantitative analysis (30).
To analyze TCRBV transcript size patterns, 24 aliquots of the cDNA were amplified for 40 cycles in 50-µl reactions. A primer specific for each of the 22 functional TCRBV subfamilies (BV1-9, BV11-18, BV20-24) (31, 32) and an unlabeled primer specific for the TCR ß-chain constant region (TCRBC) was included. The cDNAs were amplified in a Perkin-Elmer (Foster City, CA) 9600 thermocycler for 40 cycles (denaturation 25 s at 94°C, annealing 45 s at 60°C, extension 45 s at 72°C). Aliquots of the 24 PCR products were then labeled by five cycles of elongation in a "runoff" reaction with a fluorescent primer (6-carboxyfluorescein-TCRBC) (30, 33). Products of these reactions were electrophoresed on 24 cm 6% acrylamide gels on a 373 DNA sequencer and then analyzed using Genescan software (Perkin-Elmer), as previously described (30).
To quantify TCRBV transcripts, 24 aliquots of the cDNA were amplified
in 50-µl reactions as above except the primer specific for the TCRBC
was labeled with the blue fluorescent label 6-carboxyfluorescein
(34), and a primer set specific for a TCR -chain
constant region transcript (TCRAC) was added to provide an internal
control (35). The 3' TCRAC primer was labeled with a green
fluorescent label tetrachloro-6-carboxyfluorescein (TET). Thirty cycles
of PCR were used. The products were electrophoresed and analyzed for
size and fluorescence intensity as previously described (30, 36).
Sequence analysis
The BV subfamily-specific primers used in semiquantitative and
size pattern analysis were modified to contain an additional 12 base
sequences containing uracil for direct cloning into the pAMP vector
(Life Technologies) according to the manufacturer’s protocol. PCR
amplification from cDNA was conducted as described above for 40 cycles,
and 5 µl of this reaction was used to anneal into the vector. The
product was then used to transform DH5
competent cells (Life
Technologies). DNA was isolated using plasmid isolation kit (Qiagen,
Chatsworth, CA) and sequenced using FS dye terminator cycle sequencing
(Perkin-Elmer) and electrophoresed on 6% polyacrylamide gels in a
Perkin-Elmer 373 automated sequencer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical characteristics of the study patients are shown on
Table I
. For the purpose of discussion
patients were divided into four groups. Patients within group A have
been infected with HIV for at least 13 years, with stable peripheral
blood CD4+ T cell counts between 690 and 1200
cells/mm3 and typically maintain plasma virus
levels below 50 copies/ml measured by branched chain DNA (bDNA).
Patient 8 has not had viremia detected by bDNA since diagnosis until a
recent increase to 324 copies/ml associated with a febrile illness.
Patients 3–6 have shown strong proliferative responses to p24 Ag
(
cpm 9,752–27,778; stimulation index 10–140) in conventional
lymphoproliferation assays (23). Using the same assay,
similar CD4+ T cell-mediated proliferative
responses to HIV Ags have also been found in patients 7, 8, and 25
(cpm 5,997, 10,045, and 2,911, respectively) when stimulated with 8
µg/ml of p24 Ag (A. McNeil, unpublished observations). These
responses are similar to those recently described in two patients with
plasma viral RNA below 50 copies/ml plasma (11, 37). In
all other patients in the present study, the proliferative response to
p24 was <1000 cpm, which was equivalent to uninfected controls.
Patients within group B meet more commonly used clinical criteria of
nonprogressive disease (infection >7 years, peripheral
CD4+ T cell count >500 cells/µl without
antiretroviral use) (38, 39). Group C contains patients
with progressive disease not receiving antiretrovirals. Because of the
lack of availability of patients with progressive disease not receiving
antiretroviral therapy, six patients (group D) receiving therapy but
with plasma virus RNA levels >1000 copies/ml were included for
comparison. The MHC class I and class II haplotypes of the patients are
shown in Table II. HLA B*57 was
overrepresented in group A patients that are part of a larger cohort of
such patients examined in further detail in another report
(24). Only patient 25 was heterozygous for the 32-bp
deletion within the HIV coreceptor CCR5.
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The magnitude of HIV-specific CD8+ T cell
responses detected by direct CTL assays in a subset of patients was
determined. The results are shown in Fig. 1. In preliminary experiments, specific
lysis above 10% was not reproducibly observed in uninfected
individuals. Positive CTL activity was considered to be >10% specific
lysis. In preliminary experiments, differences in results obtained with
fresh or cryopreserved samples varied <10% at a given E:T ratio,
which is within the variability of this assay. These assays were
performed without restimulation of effectors and in the absence of
exogenous IL-2. The predominant activity was against gag-pol
(10–30% sp. act.), followed by nef and by env.
The level of this activity is consistent with that previously published
for patients with progressive or nonprogressive disease (Ref.
40 ; reviewed in Ref. 41).
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secretionTo further characterize the CD8+ T cell response to HIV Ags by a more quantitative method, we adapted a method of intracellular cytokine staining detected by flow cytometry to enumerate Ag-specific CD8+ T cells at the single cell level. HIV-vaccinia recombinant-infected EBV-transformed autologous B cells were used as stimuli. Similar assays have been used with peptide-pulsed target cells to measure responses to CMV in one report in humans and in virus infections in mice (16, 17, 42). Such assays have the advantage of not requiring in vitro proliferation of effector CD8+ T cells, which likely results in the low and variable numbers of these cells detected by traditional limiting dilution analysis. In addition, the determination of the response to the products of whole genes in the present study is not limited to a single peptide and permits a determination of the global response to HIV Ags.
An example of intracellular staining of stimulated cells from patient 8
is shown in Fig. 2A. Gating on
CD3+ CD8bright lymphocytes, the
percent of cells that were CD69+
IFN-+ in response to a given stimulus was
determined. No CD3+CD8+
cells were shown to synthesize IFN- after a 6-h incubation in the
absence of stimulation. When PBMC were incubated with uninfected or
vac-ß-gal-infected EBV-transformed autologous B
cells, the background percent positive cells was 6.87 for patient 5 and
more typically between 0.04 and 2 for the remaining patients. As the
percents were equivalent under these two conditions, it is likely these
cells were activated by either the B cells alone or EBV Ags
(43) and are not vaccinia specific. The response to
individual gene products is shown in Fig. 2B. The sum of the
responses to gag and pol is
equivalent to the response to the gag-pol construct gene
products. Similarly, the sum of responses to p17 and p24 is equivalent
to the gag response, indicating this method is highly
quantitative. Similarly, the percent of CD8+ T
cells that produce IFN- in response to peptide-pulsed B
cells closely correlates with the percent obtained by the corresponding
peptide MHC tetramers (not shown), consistent with recent results
obtained by an IFN- enzyme-linked immunospot assay
(44). The observed frequencies of Ag-specific cells were
also highly reproducible by this method in repeated experiments in each
of the patients studied.
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+ in
response to a given HIV gene product are shown in Fig. 3. By this method, the fraction of cells
that were HIV specific varied from 0.8 to 18.0%. Preliminary
experiments over a broad range of E:T ratios showed no higher response
to increased target numbers, only higher background activity to the
vac-ß-gal control. Similar to the direct CTL data, the predominant
activity was again directed against gag-pol with a frequency
of CD69+ IFN-+ cells
between 0.43 and 17.02%. The response in patient 8 was observed in
multiple samples taken over several months and is dramatically higher
than that observed in other patients. It is also higher than one would
predict based upon direct CTL. It is possible this response is related
to a recent increase in plasma viremia associated with a febrile
illness and may diminish with time. With the exception of the response
in patient 8, there was a good correlation between the percent of
CD8+ T cells reacting to gag-pol and
direct cytolytic activity to this target
(R2 = 0.796, p =
0.007). If the response of patient 8 is included in this correlation,
it is no longer significant (R2 = 0.3,
p = 0.15). The lack of a single-cell assay for
cytolytic activity does not permit the effector CTL function of
IFN--producing cells to be directly confirmed. It has recently been
shown in humans and in mouse models of viral infection that even memory
CD8+ T cells will rapidly secrete IFN- upon
restimulation and may remain cytolytically active (16, 45, 46). Thus, the cells detected in the current study by
intracellular cytokine staining are likely a composite of
Ag-experienced cells with either memory or effector function.
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In some virus infections in experimental animals, the number of
IFN--secreting CD8+ T cells specific for a
given peptide has been found to be similar to that detected by MHC
tetramers (16, 17). Given that such large numbers of
Ag-specific cells were found in HIV-infected patients, one concern is
that the number of activated cells detected may be increased through
bystander effects of cytokine secretion in vitro. However, the duration
(6 h) of the assay and addition of brefeldin-A to stop cytokine
secretion makes this possibility unlikely. To address these issues, we
used two well-characterized HLA-A*0201 tetramer complexes to detect
Ag-specific cells and determine the fraction able to secrete IFN-
under the current experimental conditions (18).
Four of 11 patients tested were MHC class I A*0201 positive. Of these
patients, only two had >0.1% of CD8+ T cells
specific for the conserved gag (SLYNTVATL) or pol
(ILKEPVHGV) peptide. The tetramer staining of PBMC of patients 3 and 19
is shown in Fig. 4A. No
bystander activation of these cells was observed when cells were
stimulated with any recombinant encoding non-p17 gene products.
Although no bystander activation was observed, many MHC
tetramer+ cells did not produce IFN-. Under
the experimental conditions in Fig. 4A, almost one-half of
MHC tetramer+ cells did not produce IFN- upon
stimulation. Because the frequency of SLYNTVATL-specific cells is lower
than the total responsive to the entire gag-pol gene product
(Fig. 3
), the number of target cells of patient 3 was increased to a
1:1 ratio to engage all Ag-specific cells (Fig. 4B). This
increases the numbers of cells responding but also dramatically
increases the nonspecific activity to other Ags such as to ß-gal.
Under these conditions, 73% of MHC tetramer+
cells could be induced to make IFN-. Higher numbers of responding
MHC tetramer+ cells were not observed with higher
numbers of target cells or longer stimulation at 12 or 24 h. Thus,
of all MHC-gag tetramer+ cells only a
subset produced IFN- when stimulated with the gag or
gag-pol gene product. Nearly all MHC-gag
tetramer+ cells of patients 3 or 19 were
ultimately capable of producing detectable IFN- after stimulation
with PMA/ionomycin (Fig. 3B). Surface staining was used to
further characterize the population of
CD3+CD8+ MHC
tetramer+ cells. Of unstimulated
CD3+CD8+ MHC
tetramer+ cells, 46% were
CD38+, 90% were CD27+, and
72% were CD45RA-. This result is consistent
with the vast majority of these cells being Ag-experienced memory or
effector cells (13, 18, 48).
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Large virus-specific expansions previously have been found in some
acute and chronic infections in humans (15, 43, 49, 50, 51).
Given that patients in the present study had large numbers of
Ag-specific cells, it was of interest to determine whether large
HIV-specific expansions existed within the CD8+ T
cell Ag receptor repertoire. Of the patients examined (1, 2, 3, 4, 5, 6), large
expansions within the CD8+ TCR repertoire were
found in patients 3–6. In each case, these expansions were associated
with expansion of a single-sized transcript (Fig. 5). Sequence analysis revealed that these
expansions were monoclonal. In one case (patient 3), this monoclonal
expansion made up 
30% of the circulating CD8+
T cells. In patients 3 and 6, these expansions were shown to persist
over 4 years of study. The CD4+ T cell
repertoire was also analyzed in these patients and found to be
polyclonal. No subfamily was expanded to >15%, and all size pattern
distributions were gaussian and indistinguishable from those of
uninfected individuals.
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). BV subfamily-specific Abs were used
to stain the CD8+ T cell expansions of patients
3–5. No BV6 Ab that recognized the expansion of patient 6 was found.
The percent of CD8+ T cells that stained with
BV-specific Ab correlated well with the percentages detected by
semiquantitative PCR. No HIV specificity could be found within the BV8
expansion of patient 3 and the BV5 expansion of patient 5. Although HIV
specificity was not demonstrated for most of the expansions found, the
BV5.1 expansion of patient 4 was found to be nef specific.
Some decrease in the intensity of BV5.1 Ab staining is observed on
IFN-+ cells, consistent with
activation-induced TCR down-regulation. Small numbers of
gag-pol-specific cells were also detected in this subfamily
and likely represent the lower frequency clones detected by size
pattern analysis and sequencing of BV5.1 in this patient. Although the
expansion makes up the vast majority of clones in the BV5.1 subfamily,
only 20% of the BV5.1+ cells produced IFN- in
response to the nef gene product. Higher numbers of
IFN--producing cells were not observed in response to higher numbers
of infected target cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3–15 times the number previously estimated by
enzyme-linked immunospot or tetramer analysis (18, 40, 47, 52, 53, 54). In some cases, this percent approaches the total number
of CD3+CD38+ T cells previously believed to be mostly
due to bystander activation in infected patients (21).
This study includes a unique cohort of LTNPs that has been infected
>13 years, has strong proliferative responses to HIV Ags, and
maintains plasma virus levels <50 copies/ml. Although these patients
appear to have a potent ability to restrict virus replication, this was
not associated with higher numbers of HIV-specific
CD8+ T cells measured in vitro. Similar high
levels of virus-specific CD8+ T cells were
observed in these patients as some patients with progressive infection
and 55,000 copies of viral RNA per ml of plasma or other patients with
plasma viremia poorly controlled by antiretroviral therapy. It has
previously been proposed that patients that maintain effective
restriction of HIV replication do so because of greater HIV-specific
CD8+ T cell responses. However, previous studies
have not consistently demonstrated a significant correlation between
levels of plasma virus and CD8+ T cell responses
measured in vitro (7, 8, 10, 21, 37, 55, 56). It has been
further suggested that significant correlations between plasma virus
levels and measures of HIV-specific CD8+ T cell
responses were not detected because the assays used require in vitro
stimulation, are poorly reproducible, or are not reliably quantitative.
Subsequent more quantitative analyses using MHC tetramers dramatically
increased estimates of the numbers of HIV-specific
CD8+ T cells by 10- to 100-fold and demonstrated
an inverse correlation with plasma viremia (18). In
contrast, a similar correlation was not observed in the present study.
However, these results are in agreement with one very recent report in
which the numbers of CD8+ T cells specific for
previously described HLA-A- and -B-restricted peptides detected by
enzyme-linked immunospot assays in patients with progressive HIV
infection did not correlate with plasma viral load or
CD4+ T cell count (54).
There are a number of important differences that may account for the
apparent inconsistencies between the results of the present study with
recent observations using HIV peptide MHC tetramers (18).
First, the patient population studied here is shifted toward patients
with nonprogressive infection with lower plasma virus loads. Previous
studies have identified patients with low levels of HIV-specific
CD8+ T cell responses in end stage disease
(8, 54). Conversely, low responses have also been observed
by a variety of methods in some LTNPs with very low virus loads similar
to patients 5 and 6 (8, 21, 40, 54) suggesting the
measured CD8+ T cell responses may be dependent
on virus replication in such patients. Thus, the results of such
correlations might be dramatically affected by inclusion of patients at
either extreme of HIV infection. Second, the number of Ag-specific
cells even for conserved peptide sequences is highly variable across
patients regardless of viral burden. Although four of the patients
tested are A2 positive, only patients 3 and 19 stain >0.1% of
CD8+ T cells with the SLYNTVATL-A2 tetramer,
consistent with some previous observations (40, 53).
Further, upon mapping the response to gag peptides of nine
of these patients, the number of IFN-+ cells
detected in response to a given peptide restricted to a single MHC
allele may range from 0 to 5% with no association with viral burden
(24, 54). Last, tetramer analysis alone examines the
response specific for a given MHC allele. Because of the difficulties
of mapping and production of tetramers of a given peptide, analysis is
commonly done on only the more common MHC A and B alleles. Thus, when
the global response to HIV gene products in the context of each of a
given patient’s MHC alleles is measured, considerably higher
frequencies of Ag-specific cells are found. Although these frequencies
are quite high, it is likely they still may underestimate the true
frequency of HIV-specific cells if one were to measure the total
response to the patient’s virus or cells in the lymphoid tissues.
Although large percentages of the circulating cells were specific for
HIV they were not typically concentrated within large monoclonal
expansions but rather were scattered throughout the TCR repertoire.
Overall, this is consistent with one recent report in which
gag tetramer+ cells were found to be
contained within some expanded BV subfamilies by flow cytometry
(52). Upon examination of the CD8+
TCR repertoire of the patients in the present study, some extremely
large monoclonal expansions (up to 30%) were observed. Yet in only a
minority of cases were these expansions found to be specific for the
HIV isolate tested. It is possible these expansions are in fact HIV
specific and do not react with the isolates represented by the vaccinia
recombinants used. Alternatively, they are not HIV specific and similar
to those observed in HIV-uninfected individuals (57, 58, 59, 60, 61, 62)
or are specific for other chronic virus infections such as CMV or EBV
Ags not actively expressed in transformed B cells. The lack of similar
expansions within the CD4+ T cell compartment
confirms the observed expansions in CD8+ T cells
are not due to superantigen effects nor contamination with
CD4+ T cells. An analysis of Ag-specific
CD4+ T cells in some patients in the present
study has shown that in these patients between 0.2% and 0.8% of
circulating CD4+ T cells make IFN- in response
to HIV p55 Ag (63). Taken together, these data are then
consistent with those from experimental animals showing that the
CD4+ T cell repertoire is much less prone to
large monoclonal expansions and the numbers of virus-specific
CD8+ T cells are as much as 10-fold larger than
those of CD4+ T cells (49, 61, 64, 65, 66).
Although large numbers of Ag-specific cells were in some cases detected
by either tetramer analysis or repertoire analysis, only a subset of
these cells were able to activate, as measured by CD69 staining, or
produce IFN- when stimulated through the TCR. Only approximately
one-half of the MHC tetramer+ cells of patients 3
or 19 produced IFN- in response to the appropriate HIV gene product.
It is possible that the MHC tetramer+
IFN-- cells are unable to make IFN- after
TCR engagement such as CCR7+ memory cells
(67) or alternatively noneffector Ag-specific cells
recently observed (68). However, unlike the situation in
lymphocytic choriomeningitis virus-infected mice under conditions of
CD4+ T cell depletion,
tetramer+ IFN-- cells
in the present study were able to produce cytokine upon stimulation
with PMA/ionomycin. It is also possible that these MHC
tetramer+ IFN-- cells
are a subpopulation of MHC tetramer+ cells that
are not Ag specific. This is a possibility given the avidity of the
MHC-peptide complex was increased by producing tetramers to allow
staining of Ag-specific cells. However, this is not necessarily the
case given the BV 5.1 expansion of patient 5 was nef
specific and monoclonal yet only 20% of these cells produced IFN-
even under conditions of high numbers of infected APCs. This result
suggests that the Ag-specific cells detected by MHC tetramers that do
not activate or accumulate cytokine in response to HIV gene products
may be Ag-specific cells of relatively low avidity and may have a more
limited ability to activate in response to stimulation through the
TCR.
It should be noted that conclusions regarding protective immunity based upon correlations between plasma viremia and parameters of HIV-specific immunity should be approached with some caution. Because no inverse relationship was found between plasma virus and the numbers of Ag-specific CD8+ T cells measured in vitro does not imply these cells are not important mediators of protective immunity or restriction of virus replication in some infected patients. For reasons pointed out above the relationship between the measured CD8+ T cell response and plasma viremia is likely quite complex and dependent upon virus replication and stage of disease. Further, the qualitative nature of the CD8+ T cell response may be quite different in the context of a vaccine that might induce CD4+ and CD8+ T cell responses than in HIV-infected individuals in the context of diminished CD4+ T cell help. It is now clear by more direct evidence that CD8+ T cells are important mediators of the restriction of virus replication observed in several experimental animal models of HIV infection (1, 2, 3, 4). Similarly, the cells of patients 3–8 are able to restrict autologous and challenge virus replication when engrafted into SCID-Hu animals and restriction of challenge virus replication is abrogated by CD8+ T cell depletion (23). However, this activity was not correlated with higher CD8+ T cell responses in standard assays of suppression or cytolysis. Similarly, such patients do not appear to be distinguished by higher numbers of Ag-specific CD8+ T cells than patients with progressive disease in the present study. Although high numbers of HIV Ag-specific CD8+ T cells are maintained in infected individuals with progressive disease, these appear to have a limited capacity to restrict virus replication. The important question that remains from these results and those of others is how high-level viremia persists in many patients despite such large numbers of HIV-specific CD8+ T cells. It appears that the ability of patients within group A to restrict virus replication may lie not in the number of virus-specific cells but likely in other, qualitative measures of their CD8+ T cell response that are not accounted for in traditional assays. It should be mentioned that in several models of anti-tumor responses or disruption of CD4+ T cell function during virus infection, tumor or infection is poorly controlled by CD8+ T cells in vivo yet may retain cytolytic activity detected in vitro (69, 70, 71, 72, 73, 74). In addition to CD8+ T cell avidity, other measures of the peptide targets, CD8+ T cell-derived suppressive factors, and MHC down-regulation, which may better model in vivo restriction of virus replication, are each being pursued as part of ongoing work. Further studies of such patients with very low levels of plasma virus and maintenance of strong proliferative responses may provide important clues to qualitative differences in the HIV-specific immune response that lead to effective restriction of virus replication.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ß-gal, ß-galactosidase; TCRBC, TCR ß-chain constant region transcript; TCRAC, TCR -chain constant region transcript; bDNA, branched chain DNA; LTNP, long-term nonprogressor.
Received for publication November 2, 1999. Accepted for publication April 24, 2000.
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S. Santra, J. E. Schmitz, M. J. Kuroda, M. A. Lifton, C. E. Nickerson, C. I. Lord, R. Pal, G. Franchini, and N. L. Letvin Recombinant Canarypox Vaccine-Elicited CTL Specific for Dominant and Subdominant Simian Immunodeficiency Virus Epitopes in Rhesus Monkeys J. Immunol., February 15, 2002; 168(4): 1847 - 1853. [Abstract] [Full Text] [PDF]
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D. R. Casimiro, A. Tang, H. C. Perry, R. S. Long, M. Chen, G. J. Heidecker, M.-E. Davies, D. C. Freed, N. V. Persaud, S. Dubey, et al. Vaccine-Induced Immune Responses in Rodents and Nonhuman Primates by Use of a Humanized Human Immunodeficiency Virus Type 1 pol Gene J. Virol., January 1, 2002; 76(1): 185 - 194. [Abstract] [Full Text] [PDF]
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M. R. Betts, D. R. Ambrozak, D. C. Douek, S. Bonhoeffer, J. M. Brenchley, J. P. Casazza, R. A. Koup, and L. J. Picker Analysis of Total Human Immunodeficiency Virus (HIV)-Specific CD4+ and CD8+ T-Cell Responses: Relationship to Viral Load in Untreated HIV Infection J. Virol., December 15, 2001; 75(24): 11983 - 11991. [Abstract] [Full Text] [PDF]
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T. Matano, M. Kano, H. Nakamura, A. Takeda, and Y. Nagai Rapid Appearance of Secondary Immune Responses and Protection from Acute CD4 Depletion after a Highly Pathogenic Immunodeficiency Virus Challenge in Macaques Vaccinated with a DNA Prime/Sendai Virus Vector Boost Regimen J. Virol., December 1, 2001; 75(23): 11891 - 11896. [Abstract] [Full Text]
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M. Dybul, T.-W. Chun, C. Yoder, B. Hidalgo, M. Belson, K. Hertogs, B. Larder, R. L. Dewar, C. H. Fox, C. W. Hallahan, et al. Short-cycle structured intermittent treatment of chronic HIV infection with highly active antiretroviral therapy: Effects on virologic, immunologic, and toxicity parameters PNAS, November 29, 2001; (2001) 261568398. [Abstract] [Full Text] [PDF]
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A. C. McNeil, W. L. Shupert, C. A. Iyasere, C. W. Hallahan, J. Mican, R. T. Davey Jr., and M. Connors High-level HIV-1 viremia suppresses viral antigen-specific CD4+ T cell proliferation PNAS, November 20, 2001; 98(24): 13878 - 13883. [Abstract] [Full Text] [PDF]
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S. F. Sieg, D. A. Bazdar, C. V. Harding, and M. M. Lederman Differential Expression of Interleukin-2 and Gamma Interferon in Human Immunodeficiency Virus Disease J. Virol., October 15, 2001; 75(20): 9983 - 9985. [Abstract] [Full Text] [PDF]
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J. Lieberman, P. Shankar, N. Manjunath, and J. Andersson Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection Blood, September 15, 2001; 98(6): 1667 - 1677. [Abstract] [Full Text] [PDF]
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 J. Wilkinson and F. Gotch Immune interventions: The changing face of HIV and AIDS Br. Med. Bull., September 1, 2001; 58(1): 187 - 203. [Abstract] [Full Text] [PDF]
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G. Chen, P. Shankar, C. Lange, H. Valdez, P. R. Skolnik, L. Wu, N. Manjunath, and J. Lieberman CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection Blood, July 1, 2001; 98(1): 156 - 164. [Abstract] [Full Text] [PDF]
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T. U. Vogel, T. M. Allen, J. D. Altman, and D. I. Watkins Functional Impairment of Simian Immunodeficiency Virus-Specific CD8+ T Cells during the Chronic Phase of Infection J. Virol., March 1, 2001; 75(5): 2458 - 2461. [Abstract] [Full Text]
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P. A. Goepfert, A. Bansal, B. H. Edwards, G. D. Ritter Jr., I. Tellez, S. A. McPherson, S. Sabbaj, and M. J. Mulligan A Significant Number of Human Immunodeficiency Virus Epitope-Specific Cytotoxic T Lymphocytes Detected by Tetramer Binding Do Not Produce Gamma Interferon J. Virol., November 1, 2000; 74(21): 10249 - 10255. [Abstract] [Full Text]
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M. Dybul, T.-W. Chun, C. Yoder, B. Hidalgo, M. Belson, K. Hertogs, B. Larder, R. L. Dewar, C. H. Fox, C. W. Hallahan, et al. Short-cycle structured intermittent treatment of chronic HIV infection with highly active antiretroviral therapy: Effects on virologic, immunologic, and toxicity parameters PNAS, December 18, 2001; 98(26): 15161 - 15166. [Abstract] [Full Text] [PDF]
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