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J Virol, July 1998, p. 5840-5844, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Substitutions in a Major Histocompatibility Complex
Class II-Restricted Human Immunodeficiency Virus Type 1 gp120 Epitope
Can Affect CD4+ T-Helper-Cell Function
Christine
Lekutis* and
Norman L.
Letvin
Harvard Medical School, Beth Israel Deaconess
Medical Center, Boston, Massachusetts 02215
Received 3 December 1997/Accepted 25 March 1998
 |
ABSTRACT |
It has been suggested that the inability of the immune response to
control human immunodeficiency virus type 1 (HIV-1) replication may be
due, at least in part, to the capacity of this virus to escape from
immune recognition through mutation. While there is increasing evidence
for the importance of HIV-1-specific CD4+ T cells in
containing HIV-1 spread in the infected individual, little is known
about the consequences of HIV-1 mutation on virus-specific CD4+ T-cell function. The impact of HIV-1 sequence
variation on CD4+ T-helper (Th)- cell function was
assessed with a rhesus monkey model for immune recognition of the HIV-1
envelope (Env) glycoprotein. A series of HIV-1 Env(484-496) variant
peptides were shown to retain the ability to bind to the appropriate
rhesus monkey major histocompatibility complex class II DR molecule.
Peptides bearing substitutions at position 490, however, failed to
drive the proliferation or cytokine secretion of two well-characterized
HXBc2 Env-specific rhesus monkey CD4+ Th-cell lines.
Exogenous costimulation was ineffective in complementing the ability of
the nonstimulatory peptides to induce [3H]thymidine
incorporation by these cells. Finally, HIV-1 Env(484-496) variant
peptides with substitutions at position 490 antagonized the HXBc2 Env
peptide-induced proliferative response of the CD4+ Th-cell
lines. Thus, HIV-1 variants appear to have the capacity to neutralize
the function of virus-specific CD4+ T lymphocytes.
| |
INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) rapidly mutates in an infected individual because of its high
replication rate and the infidelity of its reverse transcriptase
(11, 12, 29). The greatest sequence variation in HIV-1 is
seen in regions of the viral envelope (Env) glycoprotein. Most infected
individuals mount a vigorous immune response against HIV-1.
Nevertheless, in the absence of therapeutic interventions, high levels
of viral replication persist and profound deterioration of the immune
system usually occurs. It has been suggested that the inability of the immune response to contain this viral replication may be due to the
capacity of the mutating virus to escape immune recognition.
There is increasing evidence for the importance of CD4+ T
cells in containing HIV-1 spread in the infected individual
(28). The extent to which HIV-1 can avoid recognition by
virus-specific CD4+ T lymphocytes has not been well
defined. Early reports suggested that human HIV-1
Env (410-429)-specific CD4+ T-helper (Th)-cell clones
could recognize peptides corresponding to divergent viral isolates
(3, 31). Similarly, studies with a large panel of HIV-1
Env (428-443) variant peptides bearing empirically introduced amino
acid substitutions indicated that evasion of
H-2Ek-restricted murine Th-cell recognition was an
unlikely event (2). In contrast, the absence of recognition
of HIV-1 Env (236-251) variant peptides by human CD4+
Th-cell clones suggested that significant differences exist between ubiquitous clade B viruses (20). More recent efforts have
documented the inability of human CD4+ Th-cell clones
to recognize third-hypervariable-loop peptides, HIV-1
Env (303- 338), corresponding to heterologous viral
isolates (9, 10, 26). These reports suggest that HIV-1 may
be able to escape from CD4+ Th-cell recognition. Little
structural information, however, has been gathered to define the amino
acid substitutions in HIV-1 peptides required to alter their binding to
major histocompatibility complex (MHC) class II molecules or to
T- cell receptors (TCR). Furthermore, the functional
consequences of alterations in an HIV-1-specific CD4+
T-cell epitope for Th cells have not been thoroughly examined.
With the development of chimeric simian-human immunodeficiency viruses
containing HIV-1 env on a simian immunodeficiency virus backbone, the role of HIV-1 Env in AIDS pathogenesis can be explored with rhesus monkeys (19, 30). We previously established
HIV-1 Env-specific CD4+ Th-cell lines from plasmid
DNA-vaccinated rhesus monkeys (18). Two of these
CD4+ Th-cell lines were found to recognize the HIV-1
Env (484-496) epitope in an MHC class II DR-restricted fashion
(17). In this study, we examined the effect of substitutions
in this HIV-1-specific CD4+ Th-cell epitope on MHC
class II molecule binding and CD4+ Th-cell recognition and
function.
| |
MATERIALS AND METHODS |
Cell lines.
Rhesus monkey B lymphocytes were transformed
with a supernatant from an immortalized B-cell line (S594) productively
infected with the baboon herpesvirus Herpes papio
(23). Transformed rhesus monkey B-lymphoblastoid-cell lines
(B-LCL) were expanded and maintained in RPMI 1640 medium (GIBCO BRL,
Gaithersburg, Md.) supplemented with HEPES (25 mM),
L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (40 µg/ml), gentamicin (50 µg/ml), and 10% fetal calf serum (FCS)
(BioWhittaker, Walkersville, Md.).
HIV-1 Env-specific CD4+ Th-cell lines were established from
peripheral blood lymphocytes of HIV-1 env DNA-vaccinated
rhesus monkeys by stimulation with recombinant gp120 (Intracel,
Cambridge, Mass.) followed by expansion with recombinant interleukin 2 (IL-2) (kindly provided by Hoffmann-La Roche, Nutley, N.J.) as
previously described (18). Cells were maintained in cultures
in 24-well plates containing RPMI 1640 medium supplemented with
antibiotics and 10% FCS through repeated rounds of antigen
restimulation at 7- to 14-day intervals. The HIV-1 Env-specific Th-cell
lines were used in functional assays at least 7 days following
restimulation.
Rhesus monkey MHC class II DR
- and
-chain cDNAs were cloned into
pREP4 (Invitrogen, San Diego, Calif.) after insertion
of the
encephalomyocarditis virus internal ribosomal entry sequence
(
6). Plasmid DNA was electroporated into MHC class
II-negative,
human Epstein-Barr virus-transformed RM3 B-LCL
(
4). Stable
RM3 transfectants expressing rhesus monkey MHC
class II DR molecules
were isolated through hygromycin B (Calbiochem
Novabiochem Corp.,
La Jolla, Calif.) selection in RPMI 1640 medium
supplemented with
antibiotics and 10% FCS (
17). Cell
surface expression of MHC
class II DR molecules was confirmed by
staining with the phycoerythrin-conjugated,
HLA-DR-specific monoclonal
antibody L243 (Becton Dickinson Immunocytometry
Systems, San Jose,
Calif.) followed by flow cytometric analysis
on an EPICS XL apparatus
(Coulter Corp., Miami, Fla.).
Synthetic peptides.
The 20-amino-acid HIV-1 HXBc2 gp120
synthetic peptides used to map epitopes recognized by Env-specific
CD4+ Th-cell lines were kindly provided by the Medical
Research Council courtesy of the AIDS Reagent Project (Hertfordshire,
United Kingdom). The sequences of the peptides used in this study are
as follows: p15/Env (172-191), EYAFFYKLDIIPIDNDTTSY;
p22/Env (242-261), VSTVQCTHGIRPVVSTQLLL; and p46/Env (482-501),
ELYK Y K V V K I E PLGVAPTKA. The sequences of
the 13-amino-acid HIV-1 Env (484-496) variant peptides are listed in
Table 3. These peptides were obtained
from Quality Controlled Biochemicals (Hopkinton, Mass.).
Mamu-DR*W201-binding assay.
The binding of HIV-1
Env (484-496) variant peptides to the Macaca mulatta
leukocyte antigen (Mamu)-DR*W201 molecule expressed on the
surface of RM3 transfectants was measured in a cellular competition assay similar to that used by others (3, 33). Briefly, the ability of HIV-1 Env (484-496) variant peptides to inhibit the proliferation of a Mamu-DR*W201-restricted,
CD4+ Th-cell line specific for HIV-1 Env (172-191) was
measured. X-irradiated (5,000 rads) Mamu-DR*W201-expressing RM3 cells
were incubated in 96-well flat-bottom microtiter plates in the presence
of 25 µg of the Env (484-496) peptide competitor per ml for 2 h prior to the addition of 0.5 µg of the Env (172-191) peptide per
ml and an equivalent number of Env (172-191)-specific
CD4+ Th cells. Cultures were pulsed with
[3H]thymidine on day 2 and harvested on day 3. The
incorporation of [3H]thymidine was counted by liquid
scintillation, as described below.
Proliferation assay.
CD4+ Th-cell lines were
cultured at 2 × 104 cells/well with an equivalent
number of X-irradiated (5,000 rads) autologous B-LCL in the presence of
0 to 50 µg of peptide per ml in 96-well flat-bottom microtiter
plates. After 2 days, 1 µCi of [3H]thymidine was added
per well. The cultures were harvested onto glass fiber filter mats on
day 3 with an automated plate harvester (Tomtec, Orange, Conn.).
[3H]thymidine incorporation was determined with Optiphase
Betamix scintillation fluid and a Microbeta 1450 liquid scintillation counter (Wallac, Gaithersburg, Md.).
To reconstitute the ability of HIV-1 Env (484-496) variant peptides
bearing substitutions at position 490 to stimulate the
proliferation of
HXBc2 Env (484-496)-specific CD4
+ Th cells, 40 ng of
phorbol myristate acetate (PMA) per ml, 200
ng of ionomycin (ION) per
ml, or 100 pg of human recombinant IL-1
per ml was added to cultures
containing 5 µg of Env (484-496)
variant peptides per ml. Both PMA
and ION were obtained from Sigma
Chemical Co. (St. Louis, Mo.);
recombinant human IL-1
was purchased
from Genzyme (Cambridge,
Mass.). These studies were in all other
respects identical to the
proliferation assays already described.
Cytokine secretion.
To measure cytokine production by
CD4+ Th-cell lines, 106 T cells were cultured
with an equivalent number of X-irradiated (5,000 rads) autologous B-LCL
in 24-well plates containing 1 ml of RPMI 1640 medium supplemented with
antibiotics and 5% FCS in the presence or absence of 5 µg of peptide
per ml. Supernatants were harvested after 48 h and frozen prior to
analysis. Cytokine measurements were made according to manufacturers'
instructions with commercially available enzyme-linked immunosorbent
assay (ELISA) kits. The human IL-4 kit, the human IL-10 kit, and the
rhesus monkey gamma interferon kit were obtained from Biosource
International (Camarillo, Calif.), while the human transforming growth
factor kit and the human tumor necrosis factor alpha kit were
obtained from Genzyme. The human ELISA kits were selected on the basis
of cross-reactivity with cytokines present in the supernatants of
activated rhesus monkey peripheral blood lymphocytes.
Antagonism assay.
The ability of HIV-1 Env (484-496)
variant peptides to antagonize the proliferative response of
HXBc2-specific CD4+ Th-cell lines was examined with a
prepulse assay to eliminate peptide competition for MHC class II
occupancy (7). Autologous B-LCL were pulsed for 2 h
with 0.5 µg of the HIV-1 HXBc2 Env (484-496) index peptide per ml.
The B-LCL were then washed extensively and X-irradiated (5,000 rads)
prior to a 2-h pulse with a 0.5 to 50 µg/ml amount of the index
peptide, the Env (484-496) variant peptides, or the nonstimulatory,
Mamu-DR*W201-binding Env (172-191) peptide p15. The B-LCL were again
washed thoroughly and plated at 2 × 104 cells/well in
96-well flat-bottom microtiter plates with an equivalent number of
HXBc2 Env (484-496) peptide-specific CD4+ Th cells.
Cultures were pulsed with [3H]thymidine on day 2 and
harvested on day 3. The incorporation of [3H]thymidine
was counted by liquid scintillation, as described above.
| |
RESULTS |
Variation of HIV-1 clade B Env sequences in the region of a
CD4+ Th-cell epitope is greatest at amino acid residues
490 and 496.
We previously identified the nonamer YKVVKIEPL (amino
acids 486 to 494) as a minimal HIV-1 Env CD4+ Th- cell
epitope by using long-lived cell lines isolated from HIV-1
env DNA-vaccinated rhesus monkeys (18).
Although this CD4+ Th- cell epitope is located
in the fifth conserved region of gp120, some sequence variation has
been observed in this location, both across HIV-1 clades and among
clade B isolates (Tables 1 and 2). In
fact, when 92 clade B Env sequences were compared to the HIV-1 HXBc2
index sequence, two frequent conservative substitutions were seen:
arginine for lysine at position 490 and isoleucine for valine at
position 496 (21). These two positions appear to be relative
hot spots for viral variation.
HIV-1 Env (484-496) variant peptides retain the ability to bind
to the MHC class II molecule Mamu-DR*W201.
We also previously
showed that recognition of both HIV-1 Env (482-501) and HIV-1
Env (172-191) peptides by CD4+ Th- cell lines is
restricted by the same MHC class II DR molecule, Mamu-DR*W201
(17). It is conceivable that some Env (484-496) variant
peptides may not bind to Mamu-DR*W201 and therefore may go unrecognized
by HIV-1 HXBc2 Env (484-496)-specific, Mamu-DR*W201-restricted CD4+ Th cells. We used the Env (171-192)-specific,
Mamu-DR*W201-restricted CD4+ Th- cell line 431.08 in a
functional assay to determine whether any of the Env (484-496)
variant peptides could compete with Env (171-192) peptide binding to
a Mamu-DR*W201-expressing antigen-presenting cell. Specifically, 431.08 Th cells and an RM3 transfectant expressing Mamu-DR*W201 were
incubated in the presence or absence of Env (484-496) variant
peptides, as well as a suboptimal concentration of the Env (171-192)
peptide. As expected, the HXBc2 Env (484-496) peptide, but not an
irrelevant Env (241-262) peptide, inhibited the Env (171-192) peptide-driven proliferative response of 431.08 (Fig.
1). Interestingly, all of the
Env (484-496) variant peptides tested inhibited Env (171-192) peptide-stimulated [3H]thymidine incorporation,
suggesting that the corresponding amino acid substitutions within these
peptides were not sufficient to abrogate peptide binding to
Mamu-DR*W201.
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FIG. 1.
Peptides corresponding to naturally occurring variants
of the CD4+ Th- cell epitope Env (484-496)
compete with the Env (171-192) peptide for binding to Mamu-DR*W201
molecules expressed by RM3-transfected antigen-presenting cells.
Mamu-DR*W201-expressing RM3 cells were plated at 2 × 104 cells/ well in 96-well flat-bottom microtiter plates
and incubated for 2 h with 25 µg of the Env (484-496) variant
peptides or an irrelevant Env (241-262) peptide per ml.
Approximately 2 × 104 431.08 CD4+ Th
cells were then added to each well along with 0.5 µg of the
Env (171-192) peptide per ml. Cultures were pulsed with 1 µCi of
[3H]thymidine per well after 2 days and harvested on day
3.
|
|
HIV-1 Env (484-496) variant peptides with substitutions at
position 490 fail to drive proliferation and cytokine secretion by two
HXBc2-specific CD4+ Th- cell lines.
Proliferation
assays were then done to determine whether the Env (484-496) variant
peptides, which retained the ability to bind to the restricting MHC
class II DR molecule, were able to stimulate HIV-1 HXBc2
Env (484-496)-specific CD4+ Th cells. As shown in
Table 3, five of the nine
Env (484-496) variant peptides tested stimulated the HXBc2-specific
cell lines to proliferate. In contrast, the four Env (484-496)
variant peptides bearing substitutions at position 490 did not drive
[3H]thymidine incorporation above what was observed in
the presence of medium alone. Moreover, only Env (484-496) variant
peptides capable of stimulating a proliferative response were able to
induce the expression of the high-affinity IL-2 receptor CD25 (data not shown).
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TABLE 3.
Effects of variation in an HIV-1 Env Th- cell
epitope on the proliferative responses of MHC class
II-restricted CD4+ Th- cell lines
|
|
Since it has been shown in other systems that a peptide ligand can
trigger cytokine secretion by a T-cell population in the
absence of
proliferation, the ability of the Env (484-496) variant
peptides to
induce cytokine production was measured (
8). As
shown in
Fig.
2, only Env (484-496) variant
peptides capable of
stimulating [
3H]thymidine
incorporation were able to induce substantial gamma
interferon and
tumor necrosis factor alpha production. In addition,
the
CD4
+ Th- cell line 431.78, which produces IL-10 in
response to the
HXBc2 index peptide, also did so in the presence of the
I491V,
L494I, V497I, and V496L peptides. This cell line, however, did
not secrete IL-10 in the presence of Env (484-496) variant peptides
bearing substitutions at position 489 or 490. Neither of the
CD4
+ Th- cell lines produced IL-4 or transforming growth
factor
in response to either the HXBc2 index peptide or any of the
nine
Env (484-496) variant peptides examined (data not shown). Thus,
variation at position 490 of the HIV-1 Env (484-496) epitope
abolished
rather than modulated cytokine secretion by both of the
HXBc2-specific
CD4
+ Th- cell lines.
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FIG. 2.
Peptides corresponding to naturally occurring variants
of the CD4+ Th- cell epitope Env (484-496)
bearing substitutions at amino acid position 490 fail to stimulate
cytokine secretion by two CD4+ Th- cell lines.
Approximately 106 CD4+ Th cells were cultured
in the presence of an equivalent number of autologous B-LCL in 1 ml of
medium in the presence or absence of Env (484-496) variant peptides.
After 2 days, supernatants were harvested and frozen. Cytokine content
was later measured with commercially available ELISA kits.
|
|
Exogenous costimulation fails to restore the ability of HIV-1
Env (484-496) variant peptides bearing substitutions at position 490 to stimulate HXBc2-specific CD4+ Th- cell lines.
It
was formally possible that the K490R, K490Q, K490T, and K490E peptides,
when bound to Mamu-DR*W201, partially induced Th- cell activation.
To assess this possibility, the pharmacological agent PMA, the calcium
ionophore ION, or the cytokine IL-1 was added to cultures in an
attempt to complement any T-cell signaling ability of the
Env (484-496) variant peptides bearing substitutions at amino acid
490. PMA, ION, and IL-1, while able to enhance the proliferative
responses of the two CD4+ Th- cell lines to stimulatory
peptides, failed to induce substantial [3H]thymidine
incorporation by these cell lines in response to the K490R, K490Q,
K490T, and K490E peptides (data not shown). Thus, if the
Env (484-496) variant peptides with substitutions at position 490 induce a partial activation signal, this signal is weak at best.
HIV-1 Env (484-496) variant peptides antagonize the
HXBc2- specific proliferative responses of two
CD4+ Th- cell lines.
It has been demonstrated
in both murine and human model systems that altered peptide
ligands can inhibit the ability of wild-type peptide ligands to
activate CD4+ Th cells and CD8+ cytotoxic T
lymphocytes (13, 15). A prepulse assay was used to determine
whether the Env (484-496) variant peptides with substitutions at
position 490 could antagonize the proliferative responses of the
HXBc2-specific CD4+ Th cell lines driven by the wild-type
peptide. Specifically, autologous B-LCL were prepulsed with a
suboptimal concentration of the index peptide. The index peptide-pulsed
antigen-presenting cells were then washed and pulsed with increasing
concentrations of the Env (484-496) variant peptides for use in a
[3H]thymidine incorporation assay with HXBc2-specific
CD4+ Th cells. As demonstrated in Fig.
3, at high concentrations the K490R,
K490Q, K490T, and K490E peptides antagonized the index peptide-driven proliferative responses of HXBc2-specific
CD4+ Th cells. In contrast, another Env peptide whose
recognition was restricted by Mamu-DR*W201 had no effect on the index
peptide-specific proliferative responses.
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FIG. 3.
HIV-1 Env (484-496) variant peptides with
substitutions at position 490 antagonize the recognition of wild-type
Env (484-496) as measured in a proliferation assay. Autologous B-LCL
were prepulsed with 0.5 µg of the index HXBc2 Env (484-496)
peptide per ml for 2 h to stimulate the CD4+
Th- cell lines to the levels indicated by the horizontal broken
lines. Prepulsed B-LCL were then pulsed for an additional 2 h with
the indicated concentrations of the index peptide, the Env (484-496)
variant peptides, or the nonstimulatory, Mamu-DR*W201-binding
Env (172-191) peptide p15. Results are expressed as
[3H]thymidine incorporation in counts per minute. In the
absence of peptides, the background proliferation of both Th- cell
lines was less than 400 cpm.
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| |
DISCUSSION |
Certain structural requirements for peptide binding to MHC class
II molecules have been determined through the characterization of
naturally processed peptides eluted from purified MHC class II
complexes. As is the case for the MHC class II- associated invariant-chain peptide, the major anchor residues of the HLA-DR1, HLA-DR17, HLA-DR4, and H-2E ligands are located at relative positions 1, 4, 6, and 9 of the bound peptides (25, 32). This finding suggests that the amino acid residues of the HIV-1 Env nonamer YKVVKIEPL that are critical for binding to Mamu-DR*W201 correspond to
Y486, V489, I491, and L494 (18). The only substitutions at these presumed anchor positions in naturally occurring HIV-1 isolates are conservative ones and have little or no effect on MHC class II
binding and CD4+ Th- cell recognition in this system.
Position 5, on the other hand, is the primary TCR contact residue for
two well-characterized H-2Ek-restricted
CD4+ Th-cell epitopes (24). In fact, both a
T-cell line and a T-cell hybridoma specific for a moth cytochrome
c epitope having lysine at position 5 did not recognize
any of the 19 possible amino acid substitutions introduced at this
position into a moth cytochrome c peptide (27).
HIV-1 Env peptides synthesized with each of the four naturally
occurring amino acid substitutions for K490 at position 5 within the
rhesus monkey CD4+ Th- cell epitope were unable to
drive proliferation and cytokine secretion by two HIV-1 Env-specific
Th- cell lines in the present study. The inability of peptides with
unconservative substitutions (K490Q, K490T, and K490E) to drive T-cell
proliferation and cytokine secretion was not surprising, particularly
since these substitutions were only infrequently observed among HIV-1
clade B isolates. In contrast, the failure to tolerate the substitution
of one basic residue for another, K490R, was remarkable. The K490R
substitution has been observed in numerous HIV-1 clade B, clade D, and
clade O viruses. The exquisite sensitivity of the two rhesus
monkey CD4+ Th- cell lines to substitutions at residue
490 is consistent with the premise that lysine at position 5 is the
primary TCR contact residue of the HIV-1 Env (484-496) epitope.
The induction of different patterns of cytokine secretion by
CD4+ Th- cells following interactions with altered
peptide ligands has been documented with both human multiple sclerosis
and murine experimental autoimmune encephalomyelitis systems (22,
34). In light of the potential importance of the cytokine milieu
in AIDS immunopathogenesis and the suggested harmful effects of Th2 cytokines in this setting, it was important to determine whether HIV-1
variation may cause a shift in Th- cell cytokine secretion from a
Th1 to a Th2 profile (5). None of the HIV-1 Env (484-496) variant peptides tested, however, induced secretion of IL-4 or increased levels of IL-10 production by two HIV-1 HXBc2-specific rhesus
monkey CD4+ Th1-like cell lines. Hence, no evidence
of an altered peptide ligand-induced change in the
CD4+ Th- cell cytokine secretion profile was found in
this study.
The ability of empirically chosen altered peptide ligands to inhibit
the activation of murine CD4+ T-cell populations specific
for the model antigens hemoglobin and moth cytochrome c
has been convincingly demonstrated (24). The significance of
these observations for cellular immune responses in virus-induced
diseases, however, has been unclear. Interestingly, naturally occurring
hepatitis B virus and HIV-1 variants which are capable of inhibiting
the lysis of wild-type peptide-pulsed targets by CD8+
T-lymphocyte populations have been described (1, 16). In addition, wild-type peptide-induced activation of an HLA-DR1-restricted CD4+ Th- cell clone specific for influenza virus
hemagglutinin can be hindered by several variant peptides
(7). More recently, an altered peptide ligand corresponding
to the sequence of an HIV-1 isolate obtained from an infected
individual was shown to antagonize the cytolytic and proliferative
responses of a vaccine-induced Env-specific human CD4+
Th2-like clone (14). The ease with which antagonist
sequences were identified suggests that variant peptide antagonism of
T-lymphocyte function may be a widespread phenomenon among
virus-specific CD4+ Th cells as well as CD8+
cytotoxic T lymphocytes.
| |
ACKNOWLEDGMENTS |
We are grateful to R. E. Bachelder and M. E. Grigg for
critically reading the manuscript. The HIV-1 Env (171-192),
Env (241-262), and Env (481-502) peptides were provided by the
AIDS Reagent Project of the Medical Research Council.
This work was supported by Public Health Service grants AI20729,
AI35351, and CA50139.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Microbiology and Immunology, Stanford University Medical Center, D305 Fairchild Building, Stanford, CA 94305. Phone: (650) 723-7296. Fax:
(650) 723-6853. E-mail: clekutis{at}leland.stanford.edu.
| |
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J Virol, July 1998, p. 5840-5844, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
