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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3346-3354
Alloantigen-Stimulated Anti-HIV Activity
By
Ligia A. Pinto,
Sandra Sharpe,
David I. Cohen, and
Gene M. Shearer
From the Experimental Immunology Branch and Laboratory of Tumor Cell
Biology, National Cancer Institute, National Institutes of Health,
Bethesda, MD.
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ABSTRACT |
A number of studies have suggested that an immune response to human
leukocyte antigen (HLA) alloantigens may contribute to protection
against HIV infection. In the present study, we examined the effect of
alloantigen-stimulated cell lines obtained from peripheral blood
mononuclear cells (PBMC) of HIV-uninfected (HIV )
individuals and the soluble factors produced by these cell lines on
HIV-1 replication. Multiple in vitro restimulation with irradiated allogeneic PBMC from HIV donors resulted in the
expansion of CD8+ T-cell lines that inhibited HIV-1
replication when cocultured with either autologous or heterologous in
vitro-infected phytohemagglutinin (PHA) blasts. Supernatants from the
alloantigen-stimulated cell lines also inhibited HIV replication in
both PHA blasts and a chronically infected cell line. The
alloantigen-stimulated cell lines and the factors they produced
inhibited both T-cell-tropic (T) and macrophage-tropic (M) isolates of
HIV-1. Blocking experiments using anti-chemokine antibodies suggested
that this inhibition of HIV replication was not due to the
 -chemokines present in cocultures of cell lines with
HIV-infected blasts. These results indicate that
alloantigen-stimulation of PBMC from HIV
individuals activates CD8+ T cells that produce
soluble factor(s) that inhibit HIV replication of a wide spectrum of
HIV-1 isolates through a chemokine-independent mechanism.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
EFFORTS TO DEVELOP an effective anti-HIV
vaccine have predominantly concentrated on the development of subunit
vaccines as well as inactivated and live attenuated virus
vaccines.1,2 However, considerable evidence suggests that
immune responses to major histocompatibility complex antigens (MHC) can
contribute to protection against HIV-1 infection.3-7 The
demonstration that immunization of macaques with allogeneic cynomolgous
lymphocytes can protect against challenge infection with SIV grown in
simian cells raises the possibility that responses to foreign MHC might have implications for AIDS vaccine design.1 The role of MHC in protection against HIV infection is further supported by the observation that immunization with purified human leukocyte antigens (HLA) can protect macaques against challenge with human cell-grown virus.8,9 In addition, cellular proteins, including HLA
antigens, have been identified on free virions, and these
virus-associated antigens can be detected on the surface of infected
cells.10 Three dimensional homologies between HIV-1
envelope proteins and HLA antigens have been reported and raise the
possibility that anti-HLA responses will induce cross-reactive immune
responses to HIV envelope determinants.11-13 The recent
report that HIV-1 replication can be inhibited by
alloantigen-stimulated cells adds an additional potential protective
component to alloimmunization.14
The production of soluble anti-HIV factors by non-cytolytic
CD8+ T cells, originally described by Walker et
al,15-19 has become a topic of intense
investigation.20-22 The C-C chemokines RANTES, MIP-1 ,
and MIP-1 were recently identified as major components of an
HIV-suppressive factor produced by immortalized and primary CD8+ T cell lines.22 These chemokines suppress
infection by primary and monocyte/macrophage (M)-tropic HIV-1 isolates,
whereas T-cell-adapted viral strains appear to be resistant to the
chemokine effects.23,24
In the present study we analyzed the effect of T-cell lines generated
by stimulation with allogeneic peripheral blood mononuclear cells
(PBMC) and the alloantigen-stimulated factors they produce on the
replication of HIV-1 in human T-cell blasts and a chronically infected
cell line. Our results show that allogeneic stimulation can induce
production of anti-HIV-1 soluble factor(s) that suppress T-cell
(T)-tropic and M-tropic viral isolates and that the chemokines recently reported to inhibit HIV-1 replication are not responsible for
this antiviral effect.
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MATERIALS AND METHODS |
Cells.
Mononuclear cells were isolated by density gradient centrifugation from
heparinized peripheral blood from healthy HIV-seronegative (HIV) blood donors who were volunteers for platelets
at the National Institutes of Health Department of Transfusion
Medicine. The PBMC were washed twice in phosphate-buffered saline (PBS;
Life Technologies, Gaithersburg, MD) and were resuspended at 3 × 106/mL in RPMI 1640 (Life Technologies) supplemented with
10% heat-inactivated fetal calf serum (FCS; Hyclone, Logan, UT), 2 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL).
Viral stocks.
The primary isolates HIV-1BZ167, HIV-1CD, and
HIV-1EV were grown in human phytohemagglutinin (PHA)
blasts, as previously described.25 HIV-1Ba-L
was grown in monocyte-derived macrophages. Cell-free supernatants were
titered for p24 core Ag by enzyme-linked immunosorbent assay (ELISA;
AIDS Vaccine Program, NCI, Frederick MD).
To determine viral coreceptor usage, primary isolates
(HIV-1BZ167, HIV-1CD, and HIV-1EV)
were used to infect U87 cells stably transfected with CD4 and CCR1,
CCR2B , CCR3, CCR4, CCR5, or CXCR4. U87 cells expressing CD4 linked to
the neomycin resistance gene and individual chemokine receptors for
CCR1, CCR2B, CCR4, CCR5, or CXCR4 cloned into the pBABE-puro plasmid
were selected in 300 mg/mL G418 (GIBCO, Grand Island, NY) and 1mg/mL
puromycin (Sigma, St Louis, MO). Lines were plated in 6-well dishes and
grown for 1 to 2 days until 50% confluent before infection. Infections
were performed with 10 ng viral p24, corresponding to a multiplicity of
infection (MOI) of 2 to 5, in DMEM containing 20% fetal calf serum
(FCS), 2 mmol/L L-glutamine, and 50 mg/mL gentamicin. Cultures were
observed daily for at least 1 week and refed every other day when
samples were taken for p24 analysis. The maximum p24 level attained
during the duration of the infection was recorded. Dual-tropic viruses
were cloned by limiting dilution on U87/CXCR4 and U87/CCR5 cells
cultured in 96-well microtiter plates and determined by this system to
have approximately equivalent TCID50s, whether grown on
CXCR4 or CCR5 cells. The viruses were scored according to the p24 shed
after infection.
Generation of alloantigen-stimulated cell lines.
PBMC (3 × 106/mL) from randomly selected
HIV-uninfected individuals were incubated with a pool of irradiated (50 Gy) allogeneic PBMC (1 × 106/mL) from four unrelated
HIV-uninfected donors in tissue culture flasks (Costar, Cambridge, MA)
for 7 days at 37°C in 7% CO2 humidified atmosphere.
Seven days after the first round of stimulation, viable cells were
obtained by density gradient centrifugation and restimulated with the
same allogeneic cell pool. Three days after restimulation, media was
changed and interleukin-2 (IL-2; 10 U/mL; Boehringer Mannheim,
Indianapolis, IN) was added to the media. Five days after the fourth
weekly round of allostimulation, the cells were harvested, washed in
RPMI 1640, and used in cocultures with HIV-1-infected PHA-stimulated
blasts.
In some experiments, we depleted the alloantigen-stimulated cell lines
of CD4+ T cells using CD4 magnetic beads (Dynal, Lake
Success, NY), according to manufacturer's recommendations. After
enrichment, the population contained about 92% CD8+ T
cells and 5% CD4+ cells.
Generation of HIV-suppressive factors in vitro.
The cell lines (3 × 106 cells/mL) described above
were stimulated in vitro with the pools of irradiated allogeneic PBMC
(1 × 106 cells/mL). Supernatants were collected 2, 4, or 7 days after stimulation, filtered, and frozen at  70°C.
Their anti-HIV activity was tested on in vitro HIV-infected PHA blasts,
as described below. Unless otherwise stated, the final concentration of
supernatant used in all experiments was 50% (vol/vol).
In some experiments, PBMC (3 × 106/mL) were
stimulated with tetanus toxoid (1:400; Connaught Laboratories,
Swiftwater, PA) or with immobilized anti-CD3 monoclonal antibody
(Ortho-Biotech, Raritan, NJ; 10 µg/mL) for 5 days.
Supernatants were obtained and tested for anti-HIV activity.
HIV-1 infection of PHA blasts.
PBMC were adjusted to 2 × 106/mL cells in RPMI 1640 supplemented with 10% heat-inactivated FCS and stimulated with PHA (2 µg/mL; Sigma) for 3 days at 37°C. The PHA blasts were then washed
twice and the pellet was incubated for 1 hour with HIV-1 (at the
indicated concentrations of virus), resuspended in RPMI 1640/FCS, and
washed three times.
HIV-infected blasts (1 × 105 cells) were then
cocultured with either allostimulated cell lines (1 × 105 cells) or supernatants derived from the
alloantigen-stimulated cell lines in RPMI 1640 containing 10% FCS and
10 U/mL of IL-2 (in a total volume of 200 µL) in three or four
replicates in flat-bottom 96-well plates (Costar). Supernatants (100 µL) of these cultures were collected and inactivated with 100 µL of
2% Triton X-100 (Sigma) 3 and 6 days post-infection and frozen at
20°C, and fresh complete media or cell line supernatants
were added to each well. p24 antigen levels in these supernatants were
determined by ELISA (Coulter, Westbrook, ME).
Experiments were performed using cell lines that were derived from the
same donor cells as the PHA blasts (autologous) used for in vitro
infection, or from cell lines derived from cells obtained from an
unrelated donor (heterologous).
In some experiments, supernatants derived from allostimulated cell
lines (100 µL) were incubated for 3 days with an HIV-1 chronically
infected H9 cell line (2 × 104 cells/100 µL)
(H9/HTLVIIIMN NIH 1984; AIDS Research and Reference Reagent
Program, Rockville, MD) in flat bottom 96-well plates (Costar).
Supernatants of culture were obtained and assayed for HIV-1 p24 antigen
content as described above.
Quantitation of cytokine production after in vitro antigenic
stimulation.
Cytokine levels from the culture supernatants were determined by ELISA.
The following ELISA kits were used in this study: interferon-
(IFN-; Genzyme, Cambridge, MA), tumor necrosis factor- (TNF-;
PharMingen, San Diego, CA), IL-2 (Genzyme DuoSet, Cambridge, MA), IL-4,
IL-16, IFN- (Biosource International, Camarillo, CA), IL-10
(Genzyme), MIP-1, MIP-1, and RANTES (R&D, Cambridge, MA). All
assays were performed in duplicate according to manufacturer's instructions, and the cytokine levels were calculated by comparison to
standard curves using recombinant cytokines.
Cytokine neutralization experiments of alloantigen-stimulated
supernatants were performed using anti-MIP-1, -MIP-1, and -RANTES neutralizing antibodies (R&D; 50 µg/mL) or anti-IFN- antibody (Genzyme; 25 µg/mL) or mouse and goat isotype control antibodies (R&D; 50 µg/mL).
Flow cytometry analysis.
Immunofluorescence staining was performed by incubating 1 × 106 cells in wells of 96-well round-bottom plates in PBS
containing 1% bovine serum albumin (BSA) and 0.1% NaN3.
The cells were incubated with the relevant antibody (anti-CD3,
anti-CD4, or anti-CD8; Becton Dickinson, Mountain View, CA) and control
monoclonal antibodies directly conjugated with fluorescein
isothiocyanate (FITC) or phycoerythrin (PE) at 4°C, for 30 minutes
in the dark. After incubation, cells were washed three times with
buffer, fixed with 2% paraformaldehyde in PBS, and analyzed by
fluorescence-activated cell sorter (FACS; Becton Dickinson).
Chromium release assays.
Autologous uninfected and 3-day HIV-1BZ167-infected PHA
blasts were used as targets in a standard chromium release assay. In
some experiments, Epstein-Barr virus (EBV)-transformed B-cell lines
pulsed with the HIV-1 envelope peptides T1, T2, TH4.1, P18 MN, P18 IIIB
(5 µmol/L), or a specificity control myoglobin peptide were used as
targets, as previously described.26 Briefly,
51Cr-labeled target cells were incubated for 4 hours with
the alloantigen-stimulated cell line at several E:T ratios, and
51Cr release was determined in a -counter. The results
are presented as percent specific lysis determined by the following
equation:
where
maximum release and spontaneous release were the 51Cr
released during incubation with 2% Triton X-100 and medium alone, respectively.
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RESULTS |
Inhibition of HIV replication by allostimulated cell lines.
We investigated the effect of allostimulated cell lines obtained from
different healthy blood bank donors on HIV replication in vitro using
autologous or heterologous PHA-stimulated T-cell blasts. After six
rounds of stimulation, the cell lines were comprised of 97% of
CD3+ T cells (9% CD4+CD8;
32% CD4+CD8+; 56%
CD4CD8+ for one representative cell
line). The extracellular release of HIV-1 p24 core antigen assessed on
culture supernatants at day 3, 6, and 9 postinfection is shown in
Fig 1. The replication of the
HIV-1BZ167 isolate, which uses predominantly CXCR4 and CCR3
coreceptors (G. Blatner and D.I. Cohen, manuscript
submitted), was suppressed fivefold on day 6 in the wells of
HIV-infected blasts in the presence of allostimulated cell lines. This
antiviral activity was generated against HIV-infected PHA-activated
targets that were autologous or heterologous to the
alloantigen-stimulated cells (Fig 1A). A similar suppressive effect on
viral replication was observed when infection was performed with 172 TCID50/105 cells (data not shown). The
inhibitory effect observed on HIV-1BZ167 replication
appears to be primarily mediated by CD8+ T cells (Fig 1B),
because depletion of CD4+ T cells from
alloantigen-stimulated cell lines, using anti-CD4 coated magnetic
beads, did not appreciably alter the inhibitory effect on viral
replication observed by the same cells before depletion of
CD4+ T cells. This pattern of inhibition on viral
replication was verified in five independent alloantigen-stimulated
cell lines. Figure 2 illustrates the
spectrum of suppressive effects of four different
alloantigen-stimulated lines tested for their ability to inhibit viral
replication, 3 and 6 days after infection.
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| Fig 1.
Inhibition of HIV-1BZ167 replication by
alloantigen-stimulated cell lines. (A) PHA blasts were infected with
859 TCID50/105 cells, as described in Materials
and Methods section. Infected PHA blasts were cultured in the absence
( ) or presence of an alloantigen-stimulated cell line derived from
the same cells donor as the PHA blasts (autologous,  ) or an
alloantigen-stimulated cell line derived from an unrelated donor
(heterologous,  ). HIV-1 p24 core antigen was determined by ELISA.
The results represent means of triplicate cultures. (B)
HIV-1BZ167-infected PHA blasts (172 TCID50/105 cells) were incubated in the absence
or presence of an alloantigen-stimulated cell line before and after
depletion of CD4+ T cells, using anti-CD4-coated
magnetic beads. Results are expressed as p24 antigen production from
one experiment determined by ELISA.
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| Fig 2.
Effect of four different alloantigen-stimulated cell
lines on HIV-1 infection. PHA blasts were infected with
HIV-1BZ167 (172 TCID50/105 cells)
and cultured in the absence () or the presence () of
alloantigen-stimulated cell lines, derived from cells obtained from the
same donor as the one used to generate the blasts, at a ratio of 1:1.
The respective percentages of inhibition on days 3 and 6 of culture
were: (A) 82% and 39%; (B) 100% and 87%; (C) 100% and 99%; and
(D) 96% and 74%. The results represent means of triplicate
cultures.
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The antiviral effect observed was not due to cytolytic activity of the
effector cells against the HIV-infected targets as determined by
chromium-release assays using either HIV-infected targets
(Fig 3A) or autologous EBV-transformed
B-cell lines that were pulsed with HIV envelope peptides (Fig 3B),
previously shown to be epitopes for HIV-specific cytolytic T cells in
HIV-infected patients or in uninfected but exposed
individuals.26,27 Furthermore, the supernatants obtained
from alloantigen-stimulated cell lines did not inhibit the viability of
the infected PHA blasts (data not shown).
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| Fig 3.
Analysis of CTL responses of alloantigen-stimulated cells
using a standard 51Cr release assay. (A) Uninfected ()
or HIV-1BZ167-infected ( ) blasts were used as targets.
The targets were incubated with the effector cells at the indicated E:T
ratios for 4 hours. The results are expressed as percent specific lysis
as described in Materials and Methods. (B) EBV-transformed B-cell lines
from the same donor as the alloantigen-stimulated cell line were used
as targets. Targets were pulsed overnight with the HIV-1 envelope
peptides T1, T2, Th4.1, P18MN, P18 IIIB (5 µmol/L) and then incubated
with the alloantigen-stimulated cell line for 4 hours. The results are
represented as percent specific lysis observed at an E:T ratio of
60:1.
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To investigate whether the antiviral effect was due to a soluble
factor(s) induced by the alloantigenic stimulation, HIV-infected blasts
were incubated in the presence of supernatants obtained from
alloantigen-stimulated cell lines 2 to 7 days after stimulation (Fig 4A). These supernatants inhibited
infection in a pattern similar to that observed for the cell lines, and
the inhibitory effect was dose dependent (Fig 4B). To further address
the mechanism of inhibition of HIV-1 replication by the soluble
factor(s) generated by alloantigen stimulation, an
alloantigen-stimulated supernatant was cocultured with an
HIV-1MN chronically infected H9 cell line. Interestingly,
we observed that this supernatant inhibited the HIV-1 replication of
the chronically infected cell line, assessed by p24 antigen
determination (Fig 4C).
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| Fig 4.
Effect of supernatants from an alloantigen-stimulated
cell line on HIV-1 infection. (A) PHA blasts were infected with
HIV-1BZ167 (172 TCID50/105 cells)
and cultured in the absence () or the presence of supernatants that
were obtained 2 (), 4 (), and 7 days ( ) after cell line
stimulation with allogeneic cells. The results are expressed as mean of
triplicate cultures and are representative of one of two independent
experiments. (B) PHA blasts were infected with HIV-1BZ167
(172 TCID50/105 cells) and cultured with
different concentrations (vol/vol) of allostimulated cell line-derived
supernatant collected 4 days after stimulation. p24 antigen production
was assayed at 3 days postinfection by ELISA. The results are expressed
as mean of triplicate cultures using a representative supernatant. (C)
Effect of allostimulated cell line supernatant (1:2 dilution) on viral
replication in the HIV-1MN chronically infected H9 cell
line after 3 days of coculture. p24 antigen production was determined
by ELISA. Results represent means ± SEM of three different
experiments.
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To determine whether antigenic stimuli other than allogeneic PBMC would
generate supernatants with anti-HIV activity, we stimulated PBMC with
tetanus toxoid or with immobilized anti-CD3 and tested the antiviral
activity on HIV-1BZ167-infected PHA blasts. We found that
tetanus toxoid- or anti-CD3-stimulated supernatants did not appreciably inhibit HIV-1BZ167 replication when compared
with the effect of alloantigen-stimulated supernatants
(Table 1). Similarly, supernatants
generated by an HTLV-1 tax peptide-specific CD8+ T
cell clone28 did not inhibit HIV-1 replication, showing
that not all antigen-stimulated CD8+ T cells produce this
soluble factor(s) (data not shown). Furthermore, addition of autologous
PBMC to the HIV-1 cultures, instead of the allostimulated cell lines,
did not significantly decrease viral replication in infected cells as
compared with the cultures in the presence of the
alloantigen-stimulated cell lines or cell line supernatant (5 ± 14% and 88 ± 6% inhibition of viral replication in three
experiments, respectively).
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Table 1.
Effect of Supernatants Obtained From Several Types of
Stimulation on HIV-1 p24 Antigen Production by Infected
Lymphoblasts
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Inhibition of M-tropic and T-tropic isolates of HIV by
alloantigen-stimulated supernatants.
To test whether this suppression of HIV-1 replication would also
inhibit an M-tropic isolate, we performed experiments in which
HIV-1Ba-L was used to infect target cells. As shown in
Fig 5, the alloantigen-stimulated cell line
inhibited viral replication in PHA blasts infected by
HIV-1Ba-L by a factor of 26-fold on day 6. In fact,
allostimulated cell lines as well as their supernatants strongly
inhibited HIV-1Ba-L replication 3 days post-infection in a
similar fashion as observed for HIV-1BZ167
(Fig 6A). Addition of -chemokines at 200 ng/mL, a dose that was shown to efficiently inhibit HIV-1 replication
of several primary isolates,24 suppressed the replication
of HIV-1 Ba-L (Fig 6A), which has been previously described as a
-chemokine-sensitive isolate.22 However, the chemokines
did not inhibit replication of HIV-1BZ167 under conditions in which allostimulated cell lines and their supernatants potently suppressed p24 antigen production (Fig 6A). The antiviral
effect of an allostimulated cell line supernatant was also investigated on the viral isolates HIV-1CD (T-cell-tropic) and
HIV-1EV (M-tropic), to address the extent of its activity.
As shown in Fig 6B, this supernatant inhibited the replication of both
viral isolates to identical levels.
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| Fig 5.
Effect of an alloantigen-stimulated cell line on
HIV-1Ba-L replication. PHA blasts were infected with
HIV-1Ba-L (570 TCID50/105 cells) in
the absence () or the presence () of an allostimulated cell line.
Results represent means of triplicate cultures from one of four
experiments with similar results.
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| Fig 6.
Inhibition of replication of T-tropic and M-tropic HIV-1
isolates by alloantigen-stimulated cell lines and supernatants. (A) PHA
blasts were infected with HIV-1BZ167 ( , 172 TCID50/105 cells) or HIV-1Ba-L
(, 570 TCID50/105 cells) and cultured with
an alloantigen-stimulated cell line, its supernatant (1:2 dilution),
and the -chemokines MIP-1, MIP-1, and RANTES
(200 ng/mL of each). p24 antigen production was assayed at 3 days
post-infection by ELISA. Results are expressed as the percentage of p24
production by infected cultures in the absence of any treatment and
represent means of triplicate cultures. (B) PHA blasts were infected
with HIV-1CD () and HIV-1EV () (20 ng
p24/106 cells) and cultured with a supernatant derived from
an alloantigen-stimulated cell line collected 4 days after
alloantigenic stimulation. p24 production was assayed 3 days after
infection by ELISA. Results are expressed as the percentage of p24
production by infected cultures in the absence of allostimulated
supernatant. Results represent means ± SEM of two experiments
performed in triplicate.
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Cytokine production by alloantigen-stimulated cell lines.
To characterize the cytokine profile of the antiviral supernatants
derived from allostimulated cell lines, levels of IFN-, TNF-,
IL-2, IL-4, IL-10, and the HIV-1 suppressive chemokines MIP-1,
MIP-1, and RANTES were determined by ELISA. The results obtained
with an allostimulated cell line are shown in
Table 2. High levels of IFN- and IL-2
were produced by this cell line. IFN- is not likely to be solely
responsible for the antiviral effect, because day 7 data (Table 2) show
the lowest level of IFN- production but a high percent of inhibition
of HIV-1 replication by that same supernatant. The -chemokines,
IL-10, IL-16, and IFN- generated by allostimulation (Table 2) were
lower than the levels reported to have significant inhibitory effects
on HIV-1 replication.22,24,29-31
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Table 2.
Cytokine Levels Present in Supernatants Derived
From an Alloantigen-Stimulated Cell Line With Antiviral Activity
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Effect of neutralizing anti-cytokine antibodies on the
HIV-suppressive activity of alloantigen-stimulated cell lines and
supernatants.
To further address whether -chemokines are responsible for the
antiviral effect, a pool of monoclonal antibodies against MIP-1,
MIP-1, and RANTES were added to cocultures of HIV-infected blasts
with either allostimulated cell lines (Fig
7A) or with cell line-derived supernatants (Fig 7B). The
anti-chemokine antibodies did not reverse the suppressive effect of
cell lines on HIV-1BZ167 or HIV-1Ba-L
replication. Furthermore, the antiviral effect of the cell lines was
not altered by the presence of neutralizing anti-IFN- antibody (Fig
7A).
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| Fig 7.
Effect of neutralizing anti-cytokine antibodies on the
anti-HIV-1 activity of an allostimulated cell line and supernatant.
(A) PHA blasts were infected with HIV-1BZ167 (, 172 TCID50/105 cells) and HIV-1Ba-L
(,570 TCID50/105 cells) and cultured in the
absence or presence of an allostimulated cell line. Cultures containing
the alloantigen-stimulated cell line were also incubated with a pool of
antichemokine (anti-MIP-1, -MIP-1, and -RANTES) antibodies (50 µg/mL), anti-interferon- (25 µg/mL), or IgG control antibodies
(50 µg/mL). HIV p24 antigen was measured on day 3 (HIV-1BZ167) or day 6 (HIV-1Ba-L)
post-infection by ELISA. Results represent means of duplicate cultures
from one experiment. (B) PHA blasts infected with
HIV-1BZ167 were cultured with an allostimulated supernatant
in the presence or the absence of neutralizing anticytokine antibodies
as above. HIV-1 p24 was measured on day 3 post-infection by ELISA.
Results represent means of duplicate cultures from one of two separate
experiments with similar results.
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Similarly, anti-chemokine and anti-IFN- monoclonal antibodies did
not inhibit the suppressive activity of the supernatants derived from
the allogeneic cell line (Fig 7B). These data suggest that the soluble
antiviral factor that we are studying is not one of the three
-chemokines studied or IFN-. The blocking activity of these
antibodies was verified by chemokine or IFN--specific ELISA.
Neutralizing antibodies to -chemokines reduced the supernatant levels of RANTES by 67.6% (from 11.9 to 3.0 ng/mL), MIP-1 by 65.4%
(from 9.4 to 3.1 ng/mL), and MIP-1 by 74.7% (from 29.5 to 10 ng/mL). The addition of antibodies to IFN- reduced the levels of
IFN- from 21.3 to 5.3 ng/mL. Neutralizing anti--chemokines antibodies were also able to reverse the inhibitory effect of exogenous
-chemokines on the replication of HIV-1 Ba-L (from 77.8% inhibition
in the absence of neutralizing antibodies to 23% inhibition in the
presence of the neutralizing antibodies).
| |
DISCUSSION |
The present study has investigated the effect of alloantigen-stimulated
T-cell lines and their soluble factor(s) on HIV replication in
PHA-activated T-cell blasts. We observed anti-HIV activity in cell
lines derived from five different, randomly selected healthy (HIV-uninfected) blood bank donors. These cell lines, comprised mainly
of CD8+ T cells, as well as the supernatants derived from
these lines inhibited HIV replication of both T-tropic and M-tropic
isolates of HIV-1. This HIV-suppressive effect was not due to
allostimulated -chemokines or IFN-, which suggests that the
alloantigen-stimulated factor(s) might be similar to the
CD8-suppressive factor described by Walker et al.15
Furthermore, we showed that the allostimulated supernatants inhibited
p24 production by the HIV-1MN chronically infected H9 cell
line.
We observed a spectrum of suppressive activity among different cell
lines from different blood donors. Based on the small number of
individuals tested thus far, we were unable to determine whether
different HLA-haplotypes might be associated with different antiviral
potency. It will be important to determine whether recognition of
allogeneic HLA class I, class II, or both are necessary or optimal for
generating the anti-HIV activity. It is also noteworthy that in vitro
primary alloantigen stimulation of PBMC from one of four blood bank
donors that we tested induced factor(s) that inhibited HIV-1
replication (81.7% inhibition; L.A.P. and G.M.S., unpublished data,
December 1996). This observation raises the possibility
that cells from certain individuals might produce higher amounts of HIV
suppressive molecules following primary alloantigen exposure in vitro
and that in vivo alloimmunization may generate stronger allostimulated
anti-HIV inhibition.
No detectable CTL activity was observed against uninfected or
HIV-infected autologous PHA blasts and envelope peptide-pulsed EBV-transformed targets. These results indicate that the inhibition of
viral replication observed was not due to a cytolytic effect of the
CD8+ T cells against HIV-infected targets. The potent
antiviral effect of cell lines that were autologous to the HIV-infected
blasts excludes the possibility that this activity was mediated by
cytolytic effectors against HLA alloantigens.
The observation that anti-HIV activity is achieved not only by
allostimulated cell lines but also by supernatants further indicates
that it is unlikely that the mechanism of inhibition is mediated by
CTL. Titration of supernatants indicated that the antiviral effect
observed was concentration dependent. Although other studies have
reported anti-HIV factor production by CD8+ T cells from
HIV-infected15-17 and uninfected
individuals,20,21 these allostimulated T cells represent
the first antigen-stimulated production of -chemokine-independent
anti-HIV-1 factor(s). This HIV-suppressive effect appears to be
dependent on alloantigen stimulation because supernatants derived from
tetanus or anti-CD3-stimulated cultures as well as from an HTLV-1
tax peptide-specific CD8+ T-cell clone did not
inhibit HIV replication.
Different strains of HIV-1 were analyzed to investigate whether the
viral suppressive effect of the supernatants derived from alloantigen-stimulated cell lines extends to isolates exhibiting different cellular tropism. The fact that the supernatants contain factor(s) that inhibit T-cell-tropic and M-tropic virus is relevant in
view of the observations that M-tropic isolates of virus appear to be
more important in transmission of HIV infection,32 and predominate in the early stages of infection,33,34 whereas T-tropic isolates were reported to appear later in the spectrum of
HIV-associated disease.35,36 A change in coreceptor usage with disease progression was recently reported.37 Although
there is not an exact correlation between cell tropism and chemokine receptor usage,38 the use of isolates with different
coreceptor usage for infection in our study indicates that the
allostimulated cell line supernatants can inhibit isolates that use
predominantly CCR5 (HIV-1Ba-L and HIVEV), as
well as isolates that use CXCR4 (HIV-1CD) or preferentially
CXCR4 and CCR3 (HIV-1BZ167; G. Blatner and D.I. Cohen,
manuscript submitted). The broad spectrum of activity of
these supernatants was further supported by the observation that the
supernatants of alloantigen-stimulated cell lines inhibit p24
production by the chronically infected H9 cell line, which suggests
that these soluble factors inhibit HIV-1 at a post-entry level. Studies
are in progress to determine the mechanism and site of inhibition of
HIV-1 infection. Interestingly, studies using CD8+
antiviral factor (CAF) have shown that CD8+ cells can
inhibit both syncytium and nonsyncytium-inducing strains of
HIV.39
Analysis of cytokine levels in supernatants with HIV-suppressive
activity showed that the cytokines IL-2, IL-4, IFN- decreased in
concentration during 7 days of culture, whereas the inhibitory effects
of the supernatants remained constant. This difference in kinetics
suggests that these cytokines are not involved in the in vitro biologic
activity of the supernatants. The -chemokines RANTES, MIP-1, and
MIP-1 were also present in the alloantigen-stimulated supernatants.
It was reported that the C-C chemokines RANTES, MIP-1, and MIP-1
produced by immortalized and primary patient CD8+ T cells
inhibit in vitro HIV replication of M-tropic isolates but not
T-cell-line adapted strains.22 However, the concentrations of chemokines produced by the alloantigen-stimulated cell lines was 5- to 10-fold lower than the levels reported to be necessary to
efficiently block HIV-1 replication.22,24,40 Furthermore, the RANTES concentration was highest in the day-2 supernatant, although
the inhibitory effect of the supernatant remained high at all three
time points tested after allostimulation. Blocking experiments with
anti-MIP-1, -MIP-1, -RANTES, and -IFN- had no effect on the
antiviral effect of the supernatants or the allostimulated cell lines,
suggesting that this inhibitory effect on HIV replication is not
chemokine- or IFN--mediated. Also, addition of exogenous chemokines
at concentrations reported to inhibit HIV replication did not suppress
HIV-1BZ167 replication under conditions in which both cell
lines and their supernatants strongly inhibited viral replication.
Because this HIV strain uses as coreceptor for infection preferentially
CXCR4 and CCR3 and weakly CCR5 (G. Blatner and D.I. Cohen,
manuscript submitted) the lack of efficacy of
-chemokines on inhibition of viral replication is not surprising.
Nevertheless, at the same concentrations, chemokines inhibit
HIV-1Ba-L replication, an M-tropic strain of HIV that uses
CCR5 as coreceptor for infection,41 and these results agree
with previous published data.22 IL-10, IL-16, and IFN-
have been shown to inhibit HIV-1 infection of T cells.29-31
However, it is unlikely that these cytokines play a major role in the
antiviral activity detected in our study because the levels of these
cytokines in the supernatants of alloantigen-stimulated cultures are
several logs lower than the amount reported to suppress HIV
replication.
Preliminary analysis of specificity of the alloantigen-stimulated
supernatants indicates that these supernatants did not inhibit HTLV-1
replication, although they reduced cytomegalovirus (CMV) infection of
susceptible cell lines (L.A.P. and G.M.S., unpublished data, October 1996). This latter observation raises the possibility that allostimulated factor(s) could have a dual effect by inhibiting not only HIV, but also CMV, an AIDS-associated opportunistic infectious virus that may synergize with HIV.42
In conclusion, the present study shows that PBMC from randomly
selected, HIV-uninfected individuals can be stimulated with irradiated
PBMC from randomly selected uninfected individuals to generate
CD8+ T cell lines that produce a factor(s) that interferes
with HIV-1 replication in vitro. This factor, different from
HIV-suppressive -chemokines, inhibits T-cell-tropic and M-tropic
isolates of HIV-1, illustrating its versatility against a spectrum of
viral isolates. Studies are in progress to attempt to isolate and
identify the soluble anti-HIV factor(s) as well as to determine whether its suppressive effect extends to other viruses.
| |
FOOTNOTES |
Submitted September 4, 1997;
accepted June 22, 1998.
Address reprint requests to Gene M. Shearer, PhD, National Cancer
Institute, National Institutes of Health, Experimental Immunology Branch, Bldg 10, Room 4B36, Bethesda, MD 20892; e-mail:
ShearerG{at}exchange.nih.gov.
| |
ACKNOWLEDGMENT |
The authors thank Dr Susan Zolla-Pazner, New York University Medical
Center, New York, NY, for providing HIV-1BZ167; Dr William Biddisson, National Institutes of Health, MD, for the supernatants from
HTLV-1 tax-specific clones; and Dr Jay A. Berzofsky, National Institutes of Health, MD, for the HIV-1 envelope peptides.
| |
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Abstract
Introduction
Abstract
