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-Producing CD4+ T Cell Clones Following Immune Reconstitution on Highly Active Antiretroviral Therapy1
Departments of
* Immunology and
Medicine, University of Colorado Health Sciences Center, Denver, CO 80262; and
Epimmune, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 upon Ag
stimulation. Clones targeted eight distinct epitopes that varied in
their conservancy among HIV-1 strains, and responses were restricted by
one of three MHC II molecules. Clones showed a range of functional
avidities for both protein and peptide Ags. Additional studies
confirmed that multiple HIV-1 p24-derived epitopes were targeted by
IFN--producing CD4+ cells from subjects first treated
with HAART during advanced HIV-1 disease (median, 4.5 peptides/subject;
range, 3–6). These results suggest that in HAART-treated subjects
whose peripheral CD4+ T cell pools were once severely
depleted, the HIV-1-specific CD4+ T cell repertoire may
include a diverse array of clonotypes targeting multiple HIV-1
epitopes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HIV-1-specific CD4+ T cell responses measured in proliferation assays may be either the cause or the effect of the relatively low HIV-1 replication observed in these clinical settings. On the one hand, HIV-1-specific CD4+ T cells capable of proliferating in vitro might participate in immune control of HIV-1 replication in vivo. This would be consistent with studies of murine lymphocytic choriomeningitis virus infection, in which effective antiviral CD8+ T cell responses could not be maintained during chronic infection if CD4+ T cells were even transiently depleted (7). It would also be consistent with studies linking impairment of CMV-specific CD4+ T cell responses in bone marrow transplant recipients to reduced CMV-specific CD8+ T cell activity (8). A requirement for HIV-1-specific CD4+ T cell help to generate or maintain productive HIV-1-specific CD8+ T cell immunity would help to explain the uncontrolled viremia of most chronically infected subjects with weak HIV-1-specific CD4+ T cell responses.
On the other hand, unchecked HIV-1 replication may inhibit HIV-1-specific CD4+ T cell proliferation. This inhibitory effect has been inferred by studying CD4+ T cell responses in subjects first treated with HAART during chronic HIV-1 disease. Initial studies of subjects receiving antiretroviral therapy found that HIV-1-specific lymphoproliferation remained undetectable even as CD4+ T cell counts increased from pretreatment levels (9, 10, 11). However, several recent studies have detected HIV-1-specific CD4+ T cell proliferation in up to 50% of subjects treated with HAART during chronic HIV-1 infection (12, 13, 14, 15), suggesting that reconstitution of HIV-1-specific CD4+ T cell responses may occur with effective therapy. Consistent with this, longitudinal analysis of selected subjects who periodically stopped and restarted HAART showed that HIV-1-specific CD4+ T cell proliferative responses were only detectable during periods of viral suppression (16). The mechanisms by which HIV-1 replication might inhibit HIV-1-specific CD4+ T cell proliferative responses are unknown, but may include both depletion (17) and functional impairment (2, 15, 16) of HIV-1-specific CD4+ T cells.
By causing depletion or functional impairment of HIV-1-specific
CD4+ T cells, active HIV-1 replication may also
cause alterations in the HIV-1-specific CD4+ T
cell repertoire that viral suppression on HAART may help to correct.
Among total peripheral CD4+ T cells, abnormal
expansion or contraction of individual TCR V
subsets and specific
TCR complementarity-determining region 3 lengths within V subsets
have been associated with progressive HIV-1 infection (18, 19). Applied to the HIV-1-specific CD4+ T
cell subpopulation, a restricted TCR repertoire might be expected to
limit the diversity of HIV-1 epitopes targeted by the
CD4+ T cell response. Normalization of the total
CD4+ TCR repertoire after antiretroviral therapy
is more controversial, but has been observed in some cases of effective
viral suppression (19). Active thymic function
(20) leading to increases in naive
CD4+ T cell counts (21, 22) may
contribute to this process. Among HIV-1-specific
CD4+ T cells, repopulation with new thymic
emigrants might allow responses against a diversity of HIV-1 epitopes
to be reconstituted. However, the reconstituted repertoire of
HIV-1-specific CD4+ T cells may be unique,
because HIV-1-specific thymic emigrants generated after viral
suppression would not be as likely to encounter high levels of
replicating virus or viral Ags during priming and expansion.
Defining the repertoire of functional HIV-1-specific CD4+ T cell clonotypes and the diversity of epitope targets in treated, HIV-1-infected subjects is important for several reasons. Because CD4+ T cells may play both direct and indirect roles in controlling viral replication (7, 8, 23, 24), the repertoire diversity of the HIV-1-specific CD4+ T cell response may be an important factor in virologic control. For example, by targeting particularly conserved epitopes or a diversity of epitopes, the HIV-1-specific CD4+ T cell response might limit mutational escape from protective CD4+ T cells (25, 26). Analysis of the reconstituted HIV-1-specific CD4+ T cell repertoire may therefore suggest how well subjects who begin HAART during advanced disease may ultimately control viral replication without therapy. An understanding of the reconstituted HIV-1-specific CD4+ T cell repertoire may also help to guide adjuvant therapy for HIV-1 disease by determining which aspects of the CD4+ T cell response might be enhanced by therapeutic vaccination and other related approaches.
Unfortunately, the low response levels or frequencies of reactive cells
obtained in standard CD4+ T cell functional
assays have often impeded studies of this subject. Proliferation
assays, for instance, may not be sensitive or specific enough to
evaluate the HIV-1 Ag-specific CD4+ T cell
responses of HIV-1-infected subjects at the clonotypic level.
Similarly, HIV-1 Ag-specific, IFN--producing
CD4+ T cells have been found to make up a
relatively small percentage of all CD4+ T cells
in both untreated and HAART-treated, HIV-1-infected subjects (2, 15, 27). As a result, although HIV-specific
CD4+ T cells have been detected in subjects from
many clinical cohorts, their scarcity has prevented further dissection
of T cell responses with regard to individual clonotypes, epitope
targets, or restricting MHC molecules.
To investigate the impact of immune reconstitution on the repertoire of HIV-1-specific CD4+ T cells, we generated HIV-1 p24-specific CD4+ T cells clones from an HIV-1-infected subject who showed an HIV-1 p24 Ag-specific lymphoproliferative response after beginning HAART in the advanced stage of HIV-1 disease. The repertoire diversity and function of these clones were then characterized in detail. In addition, the p24 epitopes recognized by CD4+ T cells from additional HAART-treated subjects recovering from AIDS were determined using an ELISPOT assay on T cells expanded with Ag in vitro. The results of these studies emphasize the potential diversity of the HIV-1-specific CD4+ T cell repertoire in HIV-1-infected subjects in the setting of immune reconstitution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subject UH48 presented at the University of Colorado Hospital
Infectious Diseases Clinic with AIDS in May of 1998. His plasma HIV-1
RNA at that time was 28,390 copies/ml, and his peripheral CD4 count
nadir was 0 cells/µl. He was started on combination antiretroviral
therapy, and his plasma HIV-1 RNA subsequently declined to <400
copies/ml. At the time of this study, 22 mo into therapy, his CD4 count
had risen to 285 cells/µl, his viral load remained undetectable, and
he displayed a strong in vitro lymphocyte proliferative response to
HIV-1 p24 Ag. Based on molecular MHC class II typing, he expressed
HLA-DR1*0101 and HLA-DR1*1501. Based on serological typing, he
also expressed HLA-DQ5 and HLA-DQ1.
Additional HIV-1-infected, HAART-treated subjects were selected for investigation using the following criteria: 1) a pretreatment peripheral CD4 count nadir of <200 cells/µl, 2) an undetectable plasma HIV-1 RNA level on HAART for at least 12 mo leading up to the time of study, and 3) a CD4 count of at least 350 cells/µl at the time of study. Informed consent was obtained for all participating subjects, and the study was approved by the University of Colorado Health Sciences Center institutional review board.
Generation of p24-specific CD4+ T cell clones
PBMC were isolated from heparinized blood by density gradient
centrifugation. The frequency of HIV-1 p24-specific
CD4+ T cells in peripheral blood was first
determined by intracellular IFN- staining, as described below. PBMC
were resuspended at 5 x 106 cells/ml in
standard culture medium (RPMI 1640 (Invitrogen, Carlsbad, CA) with 10%
heat-inactivated human AB serum (Gemini Bio-Products, Woodland, CA),
and p24-specific cells were expanded by stimulation with 5 µg/ml
recombinant p24 Ag (NY5 strain; Protein Sciences, Meriden, CT) for 7
days at 37°C in a humidified 5% CO2
atmosphere. Expansion of Ag-specific cells was confirmed by
intracellular IFN- staining.
To isolate p24-specific, IFN--secreting CD4+ T
cells from this enriched culture, CD8+ cells were
depleted using CD8 microbeads (Miltenyi Biotec, Auburn, CA), and the
remaining cells were stimulated with 5 µg/ml p24 Ag and costimulatory
CD28 and CD49d mAbs (3 µg/ml; BD Biosciences, San Jose, CA).
Cryopreserved, autologous, CD3+ T cell-depleted
PBMC were added as additional APCs. After incubation at 37°C for
12 h, IFN--secreting cells were isolated by MACS as previously
described (28). Selected cells were plated at limiting
dilution in medium containing 10% human AB serum, 20 U/ml recombinant
human IL-2 (teceleukin; Hoffmann La Roche, Nutley, NJ), 0.8 µg/ml PHA
(Murex Diagnostics, Dartford, U.K.), and irradiated allogeneic PBMC
(1 x 106/ml) and B lymphoblastoid cell
lines (B-LCL; 1 x 105/ml). Viable
CD4+ T cell cultures were expanded with IL-2,
allogeneic feeder cells, and PHA stimulation every 13–15 days.
Although we did not formally show that each culture contained only one
T cell clonotype, we have referred to these cultures as clones for
simplicity.
Surface staining of p24-specific CD4+ T cell clones for
TCR V expression
Cloned cells were incubated for 30 min at 37°C with
biotinylated mAbs to human V2, V3.1, V5.1, V5.2, V6.7,
V7, V9, V12, V13.1, V13.2, V14, V17, V20,
V22, or V23 (provided by B. Kotzin). Cells were washed, incubated
with FITC-conjugated CD4 mAb (BD Biosciences) and streptavidin-PE
(Rockland, Gilbertsville, PA) for 30 min at 4°C, and analyzed on a
FACScan flow cytometer (BD Biosciences).
Intracellular cytokine staining
HIV-1 p24-specific, IFN--secreting CD4+
T cells in PBMC were identified as previously described
(15). Briefly, 106 PBMC were
incubated in standard culture medium with 3 µg/ml each of CD28 and
CD49d mAbs (BD Biosciences) and were stimulated with recombinant p24 Ag
(5 µg/ml), baculovirus control protein (2 µg/ml), or staphylococcal
enterotoxin B (1 µg/ml; Sigma-Aldrich, St. Louis, MO) as a positive
control. These cultures were incubated at a 5° slant at 37°C in a
humidified 5% CO2 atmosphere for 15 h. To
allow intracellular accumulation of cytokines, exocytosis was blocked
by the addition of brefeldin A (Golgi Plug; BD PharMingen, San Diego,
CA) after the first 5 h of incubation. Cells were then surface
stained with a Tricolor-conjugated CD4 mAb (Caltag, Burlingame, CA) for
30 min at 4°C. CD4-stained cells were washed once with PBS containing
1% BSA and fixed for 15 min at room temperature with solution A
(Caltag). Fixed cells were then permeabilized with solution B (Caltag)
and stained with a PE-conjugated anti-IFN- mAb (Caltag) for 30
min at 4°C. Cells were then washed, fixed in 1% paraformaldehyde,
and analyzed by flow cytometry. For intracellular cytokine analysis of
cultured T cells, the same protocol was followed, except that
autologous B-LCL were added during the incubation period with Ag as a
source of additional APCs.
For experiments using p24-specific CD4+ T cell
clones, a shorter time course was followed to allow detection of IL-2,
IL-4, and IL-5. Clones were stimulated with p24 Ag or control protein
in the presence of autologous B-LCL for 6 h at 37°C, and
brefeldin A was added to the stimulated cultures after 1 h. Cells
were then processed as described above, except that intracellular
cytokine stains included PE-conjugated mAbs against IFN-, IL-2
(Caltag), IL-4 (BD PharMingen), or IL-5 (BD PharMingen).
Lymphocyte proliferation assays (LPAs)
HIV-1 p24-specific CD4+ T cell clones were mixed with irradiated or mitomycin C-treated APCs in round-bottom, 96-well, polystyrene plates. Both clones and APCs were added at 5 x 104 cells/well. The Ags added included baculovirus control protein at 0.3 µg/ml, p24 Ag at 1 µg/ml, or p24 peptides at 2 µg/ml. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days. Plates were then pulsed with 1 µCi of [3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) for 10–16 h and harvested. Incorporation of 3H was counted using a Topcount beta counter (Packard BioScience, Meriden, CT).
Analysis of CD4+ T cell clone MHC restriction
Clones were stimulated in LPAs as described above with control
protein- or p24 Ag-pulsed APC lines (provided by J. Bill), including
either irradiated human B-LCL or murine fibroblast lines treated with
50 µg/ml of mitomycin C (Sigma-Aldrich). Human B-LCL used as APCs
included LG-2 (homozygous for HLA-DR1*0101), WT-8 (homozygous for
HLA-DR1*1501), and an autologous B-LCL. In some experiments this
autologous cell line was preincubated with 10 µg/ml of blocking mAb
to HLA-DR (L243; American Type Culture Collection, Manassas, VA),
HLA-DP (B7/221; American Type Culture Collection), or HLA-DQ (SPV-L3;
Zymed Laboratories, San Francisco, CA). Murine fibroblasts used as APCs
included the untransfected DAP3 line and a line transfected to express
HLA-DR1*0101 and HLA-DR
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Mapping of p24-specific CD4+ T cell clone target epitopes
Epitope-specific responses of CD4+ T cell clones were mapped in LPAs using autologous B-LCL presenting synthetic peptides based on the HXB2 strain of HIV-1 p24. Initial experiments employed a panel of 23 22-aa peptides, overlapping by 12 aa, spanning the entire p24 sequence (provided by B. Walker). Some clones were tested in additional experiments using a panel of 25 20-aa peptides, overlapping by 10 aa, spanning the same protein sequence (provided by AIDS Research and Reference Reagent Program, Rockville, MD). Additional experiments employed panels of 13-aa peptides overlapping by 12 aa (synthesized by PepScan, Lelystad, The Netherlands) spanning each 22-aa peptide targeted in initial experiments.
Determination of p24-specific CD4+ T cell clone functional avidity
Avidity for MHC/peptide was determined based on the proliferative response of each clone to titrated doses of recombinant p24 Ag or the 13-aa peptide that stimulated the clone to proliferate most strongly in epitope-mapping studies. Peptides and p24 Ag were serially diluted, then mixed with irradiated autologous B-LCL and CD4+ T cell clones. Final concentrations of whole p24 Ag ranged from 0.001–10 µg/ml (4.17 x 10-7 to 4.17 x 10-11 M). Equivalent molar amounts of 13-residue p24 peptides were used in additional assays. Each condition was tested in triplicate in a standard LPA as described above. The minimal effective dose (MED) of each Ag was calculated as the molar concentration required to stimulate 3H incorporation at least 2 times greater than and 3 SD above the incorporation observed for the unstimulated T cell clones.
HLA-DR peptide binding studies
HLA-DR1*0101-restricted peptides were tested for binding in
vitro to HLA-DR1*0101 and several other common HLA-DR alleles.
Binding assays were performed and analyzed as previously described
(29). In brief, purified human HLA-DR molecules (5–500
nM) were incubated with unlabeled HIV-1 p24 peptide and 1–10 nM
125I-radiolabeled probe peptides for 48 h.
Assays were performed at pH 7.0 for all HLA-DR alleles except
DR1*0301, the assay for which was performed at pH 4.5. HLA-DR
peptide complexes were separated from free peptide by gel filtration on
TSK200 columns, and the fraction of bound peptide was calculated as
described.
Determination of epitope conservancy
HIV-1 amino acid sequences at the positions corresponding to HXB2 p24 epitopes targeted by CD4+ T cell clones were searched on the Los Alamos HIV Sequence database at http://hiv-web.lanl.gov/content/hiv-db/EPILIGN/EPI.html. The conservancy of each epitope was calculated as the percentage of reported sequences identical with the HXB2 sequence.
ELISPOT analysis of p24 peptide-specific CD4+ T cell responses in expanded PBMC
CD4+ T cell responses in expanded PBMC to
a panel of overlapping p24 peptides were evaluated using an IFN-
ELISPOT assay as previously described (30), with some
modifications. Briefly, donor PBMC were cultured with 5 µg/ml of
recombinant p24 Ag at 37°C in 5% CO2 for 6–8
days. Cultured cells were depleted of CD8+ T
cells using CD8 microbeads and were added to 96-well,
nitrocellulose-backed plates (Millipore, Bedford, MA) previously coated
with 50 µl/well of 5 µg/ml of anti-IFN- mAb (1-D1K, mouse
IgG1; Mabtech, Nacka, Sweden). Cells were plated at 2–4.5 x
104 cells/well in 100 µl of standard culture
medium, and Ags were added in triplicate in 100 µl/well of standard
culture medium. Ags included 1 µg/ml of PHA, 0.5 µg/ml of
baculovirus control protein, 5 µg/ml of recombinant p24 Ag, or 5
µg/ml of individual 20-aa p24 peptides overlapping by 10 aa (AIDS
Research and Reference Reagent Program). Costimulatory mAb to CD28
(eBioscience, San Diego, CA) was added at a final
concentration of 1 µg/ml. Plates were incubated for 36–40 h at
37°C in 5% CO2, washed with PBS/0.05% Tween
20, and incubated at 37°C for 2 h with 2 µg/ml of biotinylated
anti-IFN- mAb (7-B6-1, mouse IgG1; Mabtech). Avidin-biotinylated
enzyme complex from the Vectastain ABC Elite kit (PK-6100; Vector
Laboratories, Burlingame, CA) was added at room temperature for 1
h, followed by the 3-amino-9-ethyl-carbazole peroxidase substrate.
Spots were counted using a dissecting microscope. The number of
IFN--secreting cells specific for each peptide was calculated by
subtracting the mean number of spot-forming cells (SFCs) in the
negative control wells from the mean number of SFCs for that peptide.
Responses were considered positive if the mean number of SFCs was 
3
SFCs/well after subtraction of the mean SFCs in the negative control
wells and >2 SD above the mean SFCs in the negative control wells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subject UH48 was selected as a model for studying the
reconstituted HIV-1-specific CD4+ T cell response
in chronically infected, HAART-treated subjects. After starting HAART
in 1998 with a peripheral CD4 count of 0 cells/µl, he showed an
increased CD4 count (285 cells/µl at the time of study) as well as a
strong lymphoproliferative response against the HIV-1 p24 Ag
(stimulation index of 19). However, after overnight p24 Ag stimulation,
p24-specific, IFN--producing CD4+ T cells in
his PBMC were too infrequent to detect by intracellular cytokine
staining (data not shown). To verify the presence of p24-specific
CD4+ T cells and to increase their numbers for
selection and cloning, PBMC were cultured in vitro with recombinant p24
Ag for 7 days. Again using p24 Ag stimulation and intracellular IFN-
staining, the frequency of p24-specific CD4+ T
cells in the expanded population was 4.15% of all
CD4+ cells (data not shown). Subsequent
experiments using CFSE labeling as a marker for cellular proliferation
showed that most cells from this subject producing IFN- after
expansion had divided at least seven times during the period of
expansion (data not shown). Therefore, the increase in frequency of
p24-specific cells was due at least in part to active division of HIV-1
p24-specific precursor cells and not solely to preferential survival of
these cells.
HIV-1 p24-specific CD4+ T cell clones were
generated from this expanded cell population by p24 Ag stimulation and
selection for IFN- production, followed by culture at limiting
dilution. Using this method, 70 CD4+ T cell
clones were obtained. These clones were screened for p24 specificity
using the donor’s autologous B-LCL to present p24 Ag in proliferation
assays or intracellular IFN- stains. Using these methods, 13 HIV-1
p24-specific CD4+ T cell clones were
identified.
HIV p24-specific CD4+ T cell clones from UH48 express a Th0 cytokine profile
To determine the cytokines produced by the
CD4+ T cell clones, each was stimulated with
autologous B-LCL presenting either control protein or p24 Ag and
stained for intracellular expression of IFN-, IL-2, IL-4, and IL-5.
Fig. 1 shows the results of this analysis
for a representative clone. The CD4high quadrants
on the right of these density plots show the cells of the
clone; the CD4low quadrants on the
left contain the autologous B-LCL. As shown in Fig. 1, A, C, E, and G, <0.3% of
CD4+ cells produced any cytokine tested after
control protein stimulation alone. After p24 Ag stimulation, by
contrast, the cells of this clone most frequently produced IFN-
(50.58%; Fig. 1B), followed by IL-2 (28.14%; Fig. 1D), IL-4 (17.71%; Fig. 1F), and IL-5 (6.47%;
Fig. 1H). Using IFN- production as a Th1 marker and
IL-4/IL-5 production as a Th2 marker, this clone was designated
Th0.
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production, all but one produced IFN- at significant
frequencies. Nine of these clones also produced IL-2, although at lower
frequencies than IFN- in all cases. More surprisingly, eight of
these clones also produced IL-4 at significant frequencies, and five of
these IL-4-producing clones also produced IL-5. Using the criteria for
Th subclass designation described above, eight of 13 clones were thus
designated Th0, whereas four were designated Th1. The one clone of 13
that failed to produce IFN- also failed to produce IL-2, IL-4, or
IL-5 and was found to be no longer active in two repeated trials in
which its p24-specific proliferation was measured simultaneously with
cytokine production.
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expression analysis defines a minimum of 6
p24-specific CD4+ T cell clonotypes from UH48
Each T cell clonotype is defined by its TCR sequence, including
characteristic TCRA and TCRB segments and unique junctional sequences.
One measure of the complexity of a T cell response is the number of
distinct TCR clonotypes it includes. To allow a preliminary estimate of
the clonotypic complexity of UH48’s HIV-1 p24-specific
CD4+ T cell response, we therefore tested each
clone against a panel of 16 mAbs specific for V family or subfamily
members. As shown in Table II, this
method identified the V regions expressed by 7 of the 13 clones. Of
these seven, three expressed V2, and one each expressed V5.1,
V5.2, V8.1, and V17. The remaining six clones expressed one or
more V regions not identifiable with the panel of Abs used. Thus, at
least six different V regions were expressed among the clones,
defining a minimum of six distinct clonotypes.
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The HIV-1 p24 epitopes targeted by UH48 clones were determined in three rounds of proliferation assays using autologous B-LCL to present p24-derived peptides. The mapping results for a representative clone are shown in Fig. 2. In the first round (Fig. 2A) the clone was stimulated with five pools of overlapping 22-aa peptides spanning the entire p24 amino acid sequence. In the second round the clone was stimulated with each individual peptide within the pool that stimulated it in round 1 (Fig. 2B). Finally, all possible 13-aa peptides within the clone’s target 22-mer were synthesized, and these peptides were used in the third round (Fig. 2C). The clone shown in Fig. 2 responded to four overlapping 13-mers; its recognition sequence is defined as the 10-aa sequence common to those four peptides (refer to Table II).
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expression could
not be determined by flow cytometry, four different epitope recognition
patterns were observed. If two clones that recognize different epitopes
are assumed to be of different clonotypes, these six clones must
represent at least four distinct clonotypes. The entire panel of 13
clones therefore represents at least 11 distinct clonotypes.
Subject UH48 expressed the MHC class II molecules HLA-DR1*0101 and
DR1*1501. He also expressed HLA-DQ5, which is linked to
HLA-DR1*0101, as well as HLA-DR51 and HLA-DQ1, which are linked to
HLA-DR1*1501. Restriction of p24-specific CD4+
T cell clones by these molecules was mapped using various HLA-matched
APC lines to present control protein or whole p24 Ag in proliferation
assays. MHC mapping results for three representative clones are shown
in Fig. 3. In the initial round of MHC
mapping, clones were stimulated using autologous B-LCL, autologous
B-LCL incubated with HLA-DR-blocking Ab, or homozygous B-LCL expressing
either the HLA-DR1*0101 or HLA-DR1*1501 haplotypes (Fig. 3, A and C). The clone shown in Fig. 3A
responded to the autologous B-LCL only in the absence of DR-blocking
Ab, indicating that it was DR restricted. It also responded to the
homozygous HLA-DR1 B-LCL, indicating that it was restricted by
HLA-DR1*0101. To confirm this, it was stimulated in a second round
using a murine fibroblast line stably expressing HLA-DR1*0101 (Fig. 3B). As expected, the clone responded specifically to p24
when presented by the transfected fibroblasts, but not when presented
by the untransfected control line. Additional clones were also
DR-restricted, but responded to homozygous HLA-DR1*1501 B-LCL,
indicating that they were restricted by HLA-DR1*1501 or the linked
HLA-DR51 allele (Fig. 3C). Finally, non-DR-restricted clones
were tested in an additional round in which autologous B-LCL were
blocked by mAbs to human MHC class I, HLA-DP, HLA-DQ, or HLA-DR. The
two clones not blocked by DR-blocking Ab were blocked only by Ab to
HLA-DQ in this second round (Fig. 3D). These clones were
thus restricted by HLA-DQ molecules.
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1*0101-expressing
murine fibroblasts, eight of these clones were restricted by
HLA-DR1*0101. Of the remaining five clones, three were restricted by
a molecule of the DR15 haplotype, including either HLA-DR1*1501 or
the linked HLA-DR51. The remaining two clones were restricted by
HLA-DQ. Because these two clones also responded to the homozygous DR1
B-LCL, which shares with UH48 expression of HLA-DQ5, but not HLA-DQ1,
these clones were probably restricted by HLA-DQ5.
HLA-DR binding affinities of HLA-DR1*0101-restricted p24
epitopes
The affinity of an antigenic peptide for binding to its MHC
restriction element may influence how well that peptide is presented by
APC. This, in turn, may influence to what extent T cells specific for
that peptide are primed and expanded in vivo. To investigate this
relationship, 13-aa p24 peptides including HLA-DR1*0101-restricted
epitope sequences recognized by six p24-specific
CD4+ T cell clones were tested for binding to
soluble, purified HLA-DR molecules in vitro. The concentration of p24
peptide required to displace 50% of a radiolabeled indicator peptide
(IC50) was determined, with lower
IC50 values indicating stronger binding.
As shown in Table III, the defined
HLA-DR1*0101-restricted p24 epitope peptides bound to their
restriction element in vitro with a range of affinities. Several bound
tightly to HLA-DR1*0101, including the two tested target peptides
for clone 4F5 (IC50 = 13.9 and 16.4 nM) and the
two for clone 6 (10.9 and 11.0 nM). These peptides were also highly
cross-reactive, binding tightly to several other HLA-DR molecules,
including HLA-DR1*0404 and HLA-DR1*0405. By contrast, several
HLA-DR1*0101-restricted peptides bound their restriction element
with relatively low affinity. For example, two peptides targeted by
clones 3G7 and 4E10 bound HLADR1*0101 with
IC50 values of 1380.3 and 2886.8 nM.
Surprisingly, these were the only peptides tested that were recognized
by more than one of the panel of clones from this subject. Therefore,
the affinities of these target peptides for binding to HLA-DR1*0101
ranged over 2 orders of magnitude, and there was no apparent
correlation between the MHC binding affinity of an epitope peptide and
the number of clones recognizing it.
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1*0101-restricted
CD4+ T cell clonesThe affinity of an epitope peptide for binding to MHC influences the overall avidity of the interaction between T cells and APC. This, in turn, helps determine the ability of the T cell to become activated by that APC and may also influence the functional properties that the clone will develop. To determine the functional avidities of these T cell clones for APC presenting their target peptides, clone responses to varying concentrations of whole p24 Ag or target 13-aa peptide were determined. Fig. 4 shows avidity determinations for two representative clones. Clone 6 (Fig. 4A) responded to molar concentrations of p24 Ag and its 13-aa peptide equally well. The MED, defined as the lowest concentration of Ag stimulating proliferation 3 SD above the background, was determined to be 4.17 x 10-7 M for both p24 and peptide in this assay. By contrast, clone 6C10 (Fig. 4B) responded more sensitively to whole p24 Ag (MED, 4.17 x 10-8 M) than to its target peptide (MED, 4.17 x 10-6 M).
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1*0101 with an IC50 of
1155.8 nM. At the same time, clone 6C10 also required 100-fold more
peptide than whole p24, even though its peptide bound HLA-DR1*0101
with a much stronger affinity of 28 nM. Sequence conservancy analysis of MHC class II-restricted p24 epitopes
The high mutation rate of replicating HIV-1 results in genetic variation among HIV-1 viral isolates, with certain regions of the HIV-1 genome more conserved than others (31). It might be reasoned that T cell responses against conserved epitopes would most effectively prevent viral immune escape. Therefore, in selecting HIV-1 sequences for use in candidate vaccines, investigators have sought immunogenic epitopes that are highly conserved among viral isolates. To determine the sequence conservancy of the epitopes identified here, clone recognition sequences were compared with published HIV-1 sequences available on the Los Alamos National Laboratories HIV-1 Sequence Database. Recognition sequences identified using the HIV-1 HXB2 tester strain were aligned with all other published isolates, and the percentages of isolates identical at those positions to the tester strain were determined.
As shown in Table V, the recognition sequences of the clones characterized here were conserved to varying degrees within clade B and among all HIV-1 isolates. Several were very highly conserved both in clade B and overall, including the DQ5-restricted target epitopes of clones 3B4 (92% in clade B, 94% overall) and 5D8 (100% in clade B, 76% overall) and the DR1-restricted target epitope of clone 4F5 (92% in clade B, 69% overall). Others werewell conserved only in clade B, including the DR1-restricted target epitopes of clones 6 (65% in clade B, 15% overall) and 8F10 (68% in clade B, 20% overall). The DR1-restricted target epitopes of clones 6C10 (27% in clade B, 7% overall) and 3G7/4E10 (11% in clade B, 20% overall) were poorly conserved both in clade B and overall.
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-producing
CD4+ T cells from additional HIV-1-infected subjects
treated with HAART during late stage disease
Given the nearly complete depletion of peripheral
CD4+ T cells in subject UH48 before treatment
with HAART, the breadth of his reconstituted HIV-1 p24-specific
CD4+ T cell repertoire was unexpected. We
therefore sought to determine whether such broad responses were common
among HAART-treated subjects with profound pretreatment depletion of
peripheral CD4+ T cells. Six additional
HAART-treated subjects who had started antiretroviral therapy during
full-blown AIDS were selected for study. These subjects had
pretreatment CD4 counts ranging from 9–180 cells/µl (median, 31.5),
but had all been receiving effective HAART for at least 5 years. PBMC
from these subjects were expanded by in vitro culture with recombinant
p24 Ag for 6–8 days, depleted of CD8+ T cells,
and stimulated in IFN- ELISPOT assays with individual overlapping
p24 peptides as described in Materials and Methods.
Significant p24 peptide-specific responses were expressed as the number
of peptide-specific, IFN--producing cells present per
106 expanded, CD8-depleted PBMC (refer to Table VI). Because expanded
CD4+ cells from four HIV-1-seronegative donors
failed to recognize any p24 peptide tested (data not shown), the
positive responses detected in HIV-1-infected subjects were probably
not the result of in vitro CD4+ T cell
priming.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. The resulting clones were analyzed for cytokine
profile, target epitope characteristics, and overall repertoire
diversity. Based on the diversity of this clonal repertoire, we used an
IFN- ELISPOT assay to determine the p24 peptides recognized by
CD4+ T cells from additional HIV-1-infected
subjects first treated with HAART during advanced HIV-1 disease.
Using p24 Ag stimulation followed by intracellular cytokine staining,
we showed that most clones produced both IFN- and IL-4. This Th0
cytokine profile (reviewed in Ref. 32) was unexpected,
given that several other studies have reported HIV-1-specific
CD4+ T cells with a predominantly Th1 profile,
including IFN-, but not IL-4. In intracellular cytokine staining
assays performed on peripheral CD4+ T cells from
HIV-1-infected, HAART-treated subjects, Pitcher et al.
(27) did not detect HIV-1 Ag-specific, IL-4-producing
CD4+ T cells, but did detect significant
frequencies of HIV-1-specific, IFN--producing
CD4+ T cells. Similarly, two published studies of
CD4+ T cell clones from HIV-1-infected subjects
emphasized the Th1 profiles of the clones obtained (23, 30). On the other hand, our results are consistent with a recent
study of HIV-1 Gag-specific CD4+ T cell clones
derived from an HIV-1-seronegative donor by in vitro immunization
(33).
These data do not establish that a significant proportion of this subject’s p24-specific CD4+ T cells produced IL-4 and IL-5 in vivo. Because clones were selected for both cytokine production and ability to expand in vitro, they might represent an insignificantly small fraction of the p24-specific Th cell population present in vivo. Alternatively, as seemingly polarized Th1 populations in other systems can be induced to express IL-4 in vitro (34), the clones studied here might have gained IL-4 and IL-5 expression only after selection. Nevertheless, as in other HIV-1-specific Th cell cloning studies (23, 30), the clones in our study were cultured without IL-4 or other Th2-skewing factors. Furthermore, not all clones produced IL-4, even though all clones were grown under the same conditions. Finally, all four Th1 clones recognized the same 22-aa p24 peptide. Because clones were stimulated in vitro with mitogen rather than specific Ag, the correlation of peptide specificity and cytokine profile suggests that cytokine profile differences among the clones might have been determined in vivo rather than in vitro.
Using overlapping p24 peptides to stimulate the clones in proliferation assays, we identified a minimum of eight epitope targets of HIV-1-specific CD4+ T cells from our study subject. Among these eight distinct epitopes, three appear to be previously unreported (HIV-1 Gag aa positions 167–176, 160–168, and 381–400). The remaining five overlap with peptides previously shown to stimulate proliferative responses in PBMC from HIV-1-infected subjects (1, 35, 36). However, only the epitope targeted by clone 6C10 (positions 133–141) has been mapped previously using peptides <20 aa (25). This epitope is also the only one shown to be restricted by the same MHC molecule as determined in our study. The results of Harcourt et al. (25) differed from ours in that the peptide used to stimulate CD4+ T cell responses in their study contained a leucine at position 138 rather than an isoleucine. A variant peptide with isoleucine at position 138 failed to stimulate the same CD4+ T cell response in their study. Because both variants bound well to the restricting HLA-DR1 molecule, the different recognition patterns of these variants in the two studies are probably due to differences in TCR specificity rather than differential epitope presentation.
Of the eight epitopes identified in this study, five were restricted by
HLA-DR1*0101. Not surprisingly, in vitro binding assays revealed
that several of these bound this MHC molecule with high affinity. The
epitopes recognized by clones 6 and 4F5 also bound other common HLA-DR
molecules with high affinity, making them potentially useful in
vaccines targeting individuals with diverse MHC II types. More
surprisingly, several of these epitopes bound to HLA-DR1*0101 with
IC50 values >1000 nM. Previous studies have
suggested that epitopes with such low affinity for presenting MHC are
poorly immunogenic in vivo (37). However, we noted that
the peptides used for in vitro binding studies were based on the
sequence of a laboratory-adapted reference strain of HIV-1. The viral
peptides that initiated priming and expansion of these clones in vivo
may have bound their restricting MHC with higher affinity than the
peptides used for in vitro studies. Finally, we found no clear
correlation between each epitope peptide’s MHC binding affinity and
its cognate clone’s functional avidity for either peptide epitope or
whole p24 Ag. This might result partly from discordance between the
binding affinity of each peptide for MHC and either its stability in
solution or the efficiency with which proteolytic processing liberates
it from whole p24 protein or a larger peptide fragment. It might also
result from unpredictable differences in TCR affinity, which would not
be completely determined by the affinity of MHC/peptide binding.
We found the clonal CD4+ T cell response to HIV-1 p24 Ag of our index subject, UH48, to be unexpectedly diverse in both the number of clonotypes it contained as well as the number of epitopes it targeted. To pursue this issue further, we determined the repertoire of p24 peptides targeted by CD4+ T cells from six additional HAART-treated subjects. These additional subjects all had CD4 counts of <200 cells/µl before initiating HAART and at least 350 cells/µl by the time of our study. Consistent with our findings in UH48, multiple p24 peptide-specific CD4+ T cell responses were detected in all additional subjects tested. Although all responded to fewer than seven p24 epitopes, the relative insensitivity of the ELISPOT assay compared with the cloning analysis performed for UH48 may partly account for this difference. In addition, the clonotypic and epitopic diversity revealed by both ELISPOT and cloning are likely to underestimate the total HIV-1-specific CD4+ T cell diversity in these subjects, because p24-specific CD4+ T cell clones primed and expanded in vivo may not all cross-react with the HXB2-based p24 peptide sequences used in vitro. Furthermore, although other studies have suggested that HIV-1 p24 Ag is a primary target of HIV-1-specific CD4+ T cells in infected subjects (1, 15), responses against other HIV-1 Ags probably also contribute to the HIV-1-specific repertoire.
The finding of a diverse p24-specific CD4+ T cell repertoire in these subjects is noteworthy first because this topic has not been extensively studied previously. Past studies of the HIV-1-specific CD4+ T cell repertoire have focused on either long term nonprogressors or subjects treated with HAART during acute HIV-1 infection. Using either lymphoproliferation assays (1) or CD4+ T cell clones (23, 30) to identify epitopes, these studies have generally identified no more than three epitope targets per subject. One study that examined the breadth of HIV-1 peptide-specific lymphoproliferation in untreated subjects with progressive disease used three peptide-specific responses as the cutoff for a broad HIV-1-specific response (38). By contrast, no subject in our study responded to fewer than three p24 peptides. This difference may be due partly to differences in methods, as the ELISPOT assay on expanded CD4+ T cells used in this study is probably more sensitive than a PBMC lymphoproliferation assay. On the other hand, Blankson et al. (14) found that HAART-treated subjects with pretreatment CD4 count nadirs <150 cells/µl were more likely to have detectable HIV-1-specific lymphoproliferative responses than HAART-treated subjects with higher nadirs. If profound pretreatment CD4+ T cell depletion contributed to strong HIV-1-specific CD4+ T cell reconstitution, it might also increase the HIV-1-specific CD4+ T cell diversity apparent in these assays.
The diversity of these p24-specific CD4+ T cell responses also leads us to question the origin of the HIV-1-specific clonotypes measured. The first and most trivial explanation for their origin is that they were primed in vitro during the 6–8 days of p24 Ag stimulation before cloning or ELISPOT analysis. We consider this unlikely because p24 peptide-specific responses could not be elicited from HIV-1-seronegative donors after identical periods of in vitro expansion. The second possibility is that all the clonotypes measured in this study were present in the peripheral blood of these subjects before treatment. Although we cannot rule this out, we consider it unlikely, given that peripheral blood CD4+ T cells were either rare or completely undetectable in all these subjects before treatment. These considerations suggest that some or all HIV-1-specific CD4+ T cell clonotypes detected in this study appeared in the peripheral blood only after viral suppression. Redistribution of pre-existing clonotypes from other compartments, particularly the lymph nodes, might contribute to the observed responses. This would be consistent with a study by Bucy et al. (39), which suggested that a global reduction in T cell activation following viral suppression allowed CD4+ T cells to be released from the lymphoid tissues into the blood. Alternatively, the HIV-1-specific CD4+ T cell clonotypes detected in this study may have been primed from naive, CD4+ thymic emigrants after HAART. Although the reduction of viral Ag levels with viral suppression might weigh against this possibility, increased lymphoproliferative responses to p24 Ag have been associated with persistence of intracellular HIV-1 mRNA in subjects receiving stable HAART with suppressed plasma viremia (40).
An important limitation of this study is that the p24-specific
CD4+ T cell repertoires of the study subjects
were not characterized before HAART treatment. As a result, we have
been unable to determine conclusively whether the diverse repertoires
observed here were primarily shaped before or after viral suppression.
However, a detailed analysis of the HIV-1-specific
CD4+ T cell repertoire before HAART would have
been difficult even if these patients had been followed in our study
before HAART. We have observed in related studies that the frequency of
all p24-specific, IFN--producing CD4+ T cells
in the peripheral blood of untreated subjects with advanced HIV-1
disease is near the lower limit of detection in our assays
(15). Although responses of similarly low frequency were
dissected further in the present study using Ag-specific expansions,
HIV-1-specific CD4+ T cells from untreated
subjects with active viral replication have generally failed to expand
in vitro (15). Attempts to overcome the suppressive
effects of viral replication on CD4+ T cell
proliferation will probably facilitate the dissection of clonotypes and
epitopes targeted in untreated subjects and may allow the repertoire
changes that occur with viral suppression to be followed directly.
Finally, our results emphasize that even subjects with advanced HIV-1 disease may retain the ability to respond to a broad array of HIV-1 epitopes after treatment. A previous study of virus-specific CD4+ T cells in humans has suggested that the virus-specific CD4+ T cell repertoire is capable of focusing on one or relatively few viral epitopes (24). For responses to genetically stable viral pathogens such as CMV, this focusing may benefit the host by allowing preferential expansion of the most protective T cell clonotypes. By contrast, for immune responses against genetically variable viruses such as HIV-1, epitope focusing might mean that the virus could escape from the CD4+ T cell response with mutations in relatively few epitopes. Although our study does not directly address the abundance of clonotypic CD4+ T cells specific for individual epitopes in the peripheral blood, it does show that the CD4+ T cell pool retains the ability to respond to multiple HIV-1-derived epitopes even after being severely depleted. The effect that this diversity of HIV-1-specific CD4+ T cell responses will have on the clinical course and pathogenesis of HIV-1 disease will need to be determined in future studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cara C. Wilson, Department of Medicine and Immunology, Division of Clinical Immunology, University of Colorado Health Sciences Center, Campus Box B-164, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: cara.wilson{at}UCHSC.edu
3 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; B-LCL, B lymphoblastoid cell line; LPA, lymphocyte proliferation assay; MED, minimal effective dose; SFC, spot-forming cell.
Received for publication August 5, 2002. Accepted for publication November 12, 2002.
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References |
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interferon-producing CD4+ T cells and HIV-1-specific lymphoproliferation in HIV-1-infected subjects with active viral replication. J. Virol. 76:5925.+ HIV-specific T cells during progression to AIDS. Blood 99:2505.This article has been cited by other articles:
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 J. J. Miles, D. Elhassen, N. A. Borg, S. L. Silins, F. E. Tynan, J. M. Burrows, A. W. Purcell, L. Kjer-Nielsen, J. Rossjohn, S. R. Burrows, et al. CTL Recognition of a Bulged Viral Peptide Involves Biased TCR Selection J. Immunol., September 15, 2005; 175(6): 3826 - 3834. [Abstract] [Full Text] [PDF]
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) or
baculovirus control protein (
). Presenting cell lines included
autologous B-LCL (Auto), a homozygous DR
) or
target peptide (
) at a range of concentrations. Results from clones
6 (A) and 6C10 (B) are shown as
examples.