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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, September 1999, p. 718-724, Vol. 6, No. 5
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Deficient Antibody-Dependent Cellular Cytotoxicity
against Human Immunodeficiency Virus (HIV)-Expressing Target Cells
in Perinatal HIV Infection
Ulrike
Ziegner,1,*
Donald
Campbell,1
Kent
Weinhold,2
Ian
Frank,3
Richard
Rutstein,1 and
Stuart
E.
Starr1
Division of Allergy, Immunology, and
Infectious Diseases, The Children's Hospital of
Philadelphia,1 and Division of
Infectious Diseases, Hospital of the University of
Pennsylvania,3 Philadelphia, Pennsylvania,
and Department of Surgery, Duke University, Durham, North
Carolina2
Received 15 March 1999/Returned for modification 7 June
1999/Accepted 9 July 1999
 |
ABSTRACT |
Peripheral blood mononuclear cells (PBMC) of human immunodeficiency
virus (HIV)-infected children, age-matched HIV-seronegative controls,
and HIV-infected asymptomatic and symptomatic adults were compared for
their ability to mediate antibody-dependent cellular cytotoxicity
(ADCC) and natural killer (NK) cell-mediated cytotoxicity against
target cells expressing HIV or herpes simplex virus (HSV) antigens.
Target cells consisted of CD4 lymphocytes purified from PBMC of
HIV-seronegative adults and incubated with the IIIB strain of HIV,
HUT78 cells chronically infected with IIIB, and HSV-infected human
fibroblasts. PBMC of asymptomatic HIV-infected adults were generally
able to lyse CD4 cells expressing HIV antigens. Direct correlation was
found between the magnitude of lysis and absolute CD4 cell counts in
these individuals. In contrast to these results, PBMC from HIV-infected
children were generally unable to lyse IIIB-expressing CD4 cells,
regardless of the children's clinical status, age, or absolute CD4
cell counts. Cells from HIV-seronegative adults and children did not
directly lyse these target cells either but, in contrast to cells of
HIV-seropositive children, were able to mediate cell lysis when serum
from an HIV-seropositive adult was added. However, effector cells from
these HIV-infected children were able to mediate both ADCC against
HSV-infected fibroblasts and NK cell-mediated cytotoxicity against
IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically
HIV-infected children to mediate ADCC against HIV antigen-expressing
CD4 cells may contribute to rapid progression to AIDS.
| |
INTRODUCTION |
CD4 lymphocytes are known to be
targets for human immunodeficiency virus (HIV) infection in vivo.
Therefore, the use of HIV antigen-expressing CD4 lymphocytes as target
cells in cytotoxicity assays might yield data closely reflecting in
vivo events. Previous reports indicated that peripheral blood
mononuclear cells (PBMC) of HIV-infected adults are able to lyse CD4
lymphocytes expressing gp120, the major envelope glycoprotein of HIV
type 1 (HIV-1) (55, 57, 62). The effector cells responsible
for this lysis were shown to be CD16+ natural killer (NK)
cells, armed in vivo with cytophilic HIV-specific antibodies.
Therefore, this mechanism of cytotoxicity can be classified as
antibody-dependent cellular cytotoxicity (ADCC).
To explore possible mechanisms for accelerated disease progression in
some perinatally HIV-infected children compared to that in adults
(2, 4, 49), we studied ADCC against HIV-1-expressing CD4
lymphocytes in children at various stages of HIV infection. Responses
were compared to those of HIV-infected adults and HIV-seronegative age-matched controls.
| |
MATERIALS AND METHODS |
Subjects.
Subjects consisted of HIV-infected adults,
monitored at the Hospital of the University of Pennsylvania; children
with perinatal HIV infection, monitored in the Special Immunology
Clinic at The Children's Hospital of Philadelphia; and age-matched
HIV-seronegative healthy volunteers. HIV infection was diagnosed on the
basis of at least two positive PCRs and PBMC cultures for HIV.
According to Centers for Disease Control and Prevention (CDC) criteria
for children and adults (8, 9), HIV-infected children were
classified as asymptomatic with normal (P1-A) or abnormal (P1-B) immune
function or as symptomatic with nonspecific findings (P2-A) or
HIV-related conditions (P2-B-F), whereas adults' stages were
classified as asymptomatic (CDC stage A), symptomatic conditions (stage
B), or AIDS-defining conditions (stage C). Patients receiving
intravenous immunoglobulin were excluded from the study, since repeated
administration of intravenous immunoglobulin may lead to reduced NK
cell-mediated cytotoxicity (11) and might affect the ability
of PBMC to mediate ADCC. This study was approved by the Institutional
Review Boards of the University of Pennsylvania and The Children's
Hospital of Philadelphia.
Effector cells.
PBMC were separated from heparinized venous
blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) gradient
centrifugation. Monocytes were removed by adherence on plastic surfaces
coated with fetal bovine serum (FBS; HyClone, Logan, Utah) as
previously described (23). PBMC were used in cytotoxicity
assays within 4 h after the blood drawing.
Experiments in which NK cells were depleted from PBMC by incubation
with monoclonal antibody anti-Leu 11B (Becton Dickinson, Mountain View,
Calif.), which reacts with the Fc
III receptor (CD16) on NK cells, as
previously described (3, 39) followed by incubation with
baby rabbit complement (Cedarlane Laboratories, Hornby, Ontario,
Canada) to destroy antibody-bound cells were performed. The surviving
PBMC were used as effector cells in cytotoxicity assays. Arming of
effector cells was accomplished by incubating PBMC for 12 h at
37°C with undiluted heat-inactivated heterologous sera obtained from
HIV-infected patients and seronegative controls (58). The
cells were washed five times before use as effector cells in
cytotoxicity assays. To elute putative cytophilic antibodies, freshly
isolated PBMC were incubated at 37°C for 12 h and then washed
three times (57).
Target cells.
HUT78 cells, derived from a CD4+
lymphoblastoid T-cell line, uninfected and chronically infected with
the HIV-1 strain IIIB (16), were kindly provided by J. A. Hoxie, Hospital of the University of Pennsylvania, Philadelphia.
K562 cells, derived from an erythroleukemia cell line and known to be
sensitive to NK cell-mediated cytotoxicity, were used as target cells
in NK cell assays. FS4 cells, human embryonic foreskin fibroblasts
(National Institute of Allergy and Infectious Diseases, Bethesda, Md.),
were inoculated with the NS strain of herpes simplex virus type 1 (HSV-1) (kindly provided by H. M. Friedman, Hospital of the
University of Pennsylvania) at a multiplicity of infection of 5.0, as
previously described (37). After 6 h of incubation at
37°C in 5% CO2, the cells were trypsinized, washed, and
then stored in the vapor phase of liquid nitrogen. Uninfected FS4 cells
were prepared simultaneously.
PBMC of healthy seronegative adults were stimulated with
phytohemagglutinin (Sigma, St. Louis, Mo.) and then expanded in the presence of human interleukin 2 (IL-2; Schiapparelli, Fairfield, N.J.)
as described previously (56). Thereafter, CD4 cells were selected from these phytohemagglutinin-IL-2-stimulated PBMC by panning
(63) with the monoclonal antibody OKT4. Purified
CD4+ cells were incubated for 48 h in medium
containing 32 U of IL-2 per ml and 20% FBS. The cells were then washed
and subjected to low-speed centrifugation. Cell-free supernatant of
strain IIIB-infected HUT78 cells was added to pelleted CD4+
cells at a final dilution of 1:10,000. After 1 h of incubation at
37°C, the cell surface expression of viral antigens was confirmed by
flow cytometric analysis after immunofluorescent staining with HIV-seropositive human serum and fluorescein-conjugated goat
F(ab)2 anti-human immunoglobulin G (IgG) (TAGO, Burlingame,
Calif.). After virus inactivation with 4% paraformaldehyde in
phosphate-buffered saline, viral antigen expression was quantified by
flow cytometry. Unexposed and HIV-coated target cells were
electronically gated to exclude aggregates and nonviable cells from
evaluation. Fluorescence intensity thresholds of less than 2% positive
cells were established by using uninfected target cells incubated with
HIV-1 antibody-positive human serum and HIV-1-infected target cells
incubated with HIV-1 antibody-negative human serum. HIV antigens were
detected on >90% of target cells after 1 h of exposure to
IIIB-containing supernatant.
NK cell-mediated cytotoxicity.
Target cells, consisting of
HUT78 cells chronically infected with the IIIB strain of HIV-1
(HUT78/IIIB), uninfected HUT78 cells, and K562 cells were labeled with
Na251CrO4 (Amersham, Arlington
Heights, Ill.), resuspended in RPMI containing 20% FBS, and aliquoted
into round-bottomed 96-well microtiter plate wells. Effector cells,
prepared as described above, were added to give effector/target (E:T)
cell ratios of 100:1 (5 × 105 PBMC/5,000 targets) in
a 200-µl total volume per well. In preliminary experiments with PBMC
from HIV-infected adults, maximal levels of NK cell-mediated lysis were
detected at this E:T ratio. Owing to small volumes of blood obtainable
from children, multiple E:T ratios could not be tested. After 18 h
of incubation, 100 µl of supernatant per well was harvested without
disturbing the cell pellet. Supernatants were autoclaved to inactivate
HIV before being counted in a gamma scintillation counter. All tests
were done in triplicate. Total release (100%) was determined by
addition of 100 µl of 2% Triton X-100 to target cells in the absence
of effector cells. Spontaneous release, determined by adding 100 µl
of medium instead of effector cells, was always less than 30%. Percent
51Cr release was calculated by the standard formula.
ADCC against HIV-expressing CD4 lymphocytes.
HIV-expressing
and control CD4 lymphocytes, obtained as described above, were labeled
with 51Cr, resuspended in RPMI 1640 containing 20% FBS and
32 U of IL-2 per ml, and added to PBMC to give final E:T ratios of
100:1. Microtiter plates were centrifuged (100 × g for
3 min) and incubated for 4 h at 37°C in 5% CO2.
51Cr release in supernatants was determined as described above.
ADCC against HSV-infected FS4 cells.
HSV-infected and
uninfected FS4 cells were labeled with 51Cr, resuspended in
RPMI containing 20% FBS, and added to effector cells to give E:T
ratios of 100:1. The effector cells were added to wells in medium that
contained sera from HSV-seropositive or -seronegative individuals at
final dilutions of 1:100, previously found to be the optimal
concentration for maximal ADCC-mediated killing of HSV-infected cells.
After incubation for 4 h at 37°C, 51Cr release in
supernatants was determined as described above.
Quantitation of NK cells.
Immunophenotyping was performed by
standard procedures. Heparinized venous blood was incubated with the
monoclonal antibody B73.1 (kindly provided by G. Trinchieri, Wistar
Institute, Philadelphia, Pa.), which reacts with the FcRIII (CD16)
receptor present on NK cells and polymorphonuclear leukocytes
(39). HIV was inactivated by incubation in 4%
paraformaldehyde in phosphate-buffered saline for 16 h at 4°C
prior to flow cytometric analysis. Mononuclear lymphocytes were
electronically gated to exclude other leukocytes from evaluation. After
accumulation of 5,000 events, a fluorescence intensity threshold of
less than 2% positive cells was established by using cells incubated
with control mouse IgG.
Statistical methods.
The data shown in the figures represent
means ± standard errors. The two-sample t test was
used to compare mean ADCC antibody titers for HIV-1-infected and
uninfected infants. n refers to the total number of
determinations for a particular condition.
| |
RESULTS |
Cell-mediated cytotoxicity against HIV-expressing CD4 cells.
Forty-six children, 3 months to 10 1/2 years of age, with perinatally
acquired HIV infection (19 patients at CDC classification stage P1, 15 at stage P2-A, and 12 at stage P2-B-D), were tested for cytotoxic
activity against HIV-expressing CD4 cells. As shown in Fig.
1, PBMC from 31 of 46 children failed to
lyse HIV-1-expressing CD4 cells, and PBMC of only four children gave
>3% lysis. Even for these four, corresponding levels of lysis of
control CD4 cells were not significantly different (data not shown).
The lack of cytotoxic activity against HIV-expressing CD4 cells did not
correlate with the patients' stage of infection, age, or absolute CD4
lymphocyte counts.
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FIG. 1.
Cytotoxicity against IIIB-coated CD4 lymphocytes
mediated by PBMC of HIV-infected pediatric patients at different stages
of disease: P1 ( ), P2-A ( ), and P2-B-D ( ), according to the
CDC classification for pediatric HIV infection.
|
|
Concurrently with pediatric samples, we tested the ability of PBMC
isolated from 45 HIV-seropositive adults to lyse HIV-expressing CD4
cells (Fig. 2). Most of these patients
were male homosexuals; 28 were asymptomatic (CDC classification stage
A), and 17 were symptomatic (CDC stage B or C). In contrast to the
results obtained for HIV-seropositive children, cytotoxicity was
observed for the majority of adults tested. The magnitude of target
cell lysis correlated positively with absolute CD4 cell counts: the
highest magnitude of cytotoxicity was observed when the CD4 cell count exceeded 500/mm3 (mean, 16.3%; range, 5.1 to 36.9%). PBMC
of patients with CD4 counts between 200 and 500 cells/mm3
and those with less than 200 cells/mm3 exhibited lower
cytotoxic activity (mean cytotoxicity of 9.1 and 5.6%, respectively).
Mean cytotoxicity mediated by PBMC of patients with absolute CD4 cell
counts of >500 cells/mm3 was significantly higher than
that of patients whose CD4 cell counts were <200 cells/mm3
(P < 0.05). Cytotoxicity was not detected against
uninfected CD4 cells (data not shown). PBMC of HIV-seronegative adults
(n = 30) and children (n = 21) failed
to lyse HIV-expressing CD4 lymphocytes (data not shown).
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FIG. 2.
Cytotoxicity against IIIB-coated CD4 lymphocytes,
mediated by PBMC of HIV-infected adults at different stages of disease:
asymptomatic () and symptomatic ().
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|
Characterization of effector cells and requirement for HIV-specific
antibodies.
A series of experiments was carried out to investigate
the mechanism of cytotoxicity against HIV-expressing CD4 lymphocytes in
adults. CD16+ NK cell-depleted effector cells were tested
for their ability to lyse a variety of target cells in 51Cr
release assays (Table 1). After depletion
of CD16+ cells, specific 51Cr release from all
target cells, except from uninfected CD4 lymphocytes, was significantly
decreased (P < 0.05). When the effector cells were
treated with complement alone prior to being added to cytotoxicity assays, only a slight loss of lytic activity was observed. These experiments confirm that lysis of HIV-expressing CD4 cells was mediated
primarily by NK cells.
After incubation for 12 h at 37°C, PBMC of HIV-seropositive
adults lost their ability to lyse HIV antigen-expressing CD4 cells (Table 2). The ability of such PBMC to
lyse HIV-expressing CD4 cells was restored when autologous HIV
antibody-containing serum was added to assays at a final concentration
of 1:100. These findings indicate that decreased cytotoxicity against
HIV-expressing CD4 cells after incubation of PBMC is due to elution of
cytophilic antibodies from NK cells rather than to defective effector
cells. Thus, our data confirm a previous report that lysis of
HIV-expressing CD4 lymphocytes occurs via an antibody-dependent
mechanism, mediated by NK cells armed in vivo with cytophilic HIV
antibodies (62).
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TABLE 2.
Effect of incubation on the ability of PBMC to mediate
lysis in the absence and presence of autologous serum
|
|
Mechanism and specificity of the deficient cytotoxicity against
HIV-expressing CD4 cells in HIV-infected children.
To determine
whether the inability of PBMC from perinatally HIV-infected children to
lyse HIV-expressing CD4 cells was due to a reduced number of effector
cells, we used flow cytofluorography to determine the
percentage of NK cells in PBMC populations. Seventeen HIV-infected
pediatric patients, 14 weeks to 8 years of age (stage P1 to P2-D1) had
NK cell percentages within the normal range for age-matched
seronegative children (range, 11.5 to 22.8%; mean, 17.6%).
To determine whether decreased cytotoxic activity of PBMC from
perinatally HIV-infected children was specific for HIV-expressing CD4
cells, we tested PBMC of HIV-infected and uninfected children and
adults for their ability to lyse FS4 fibroblasts, infected with HSV-1
strain NS, as previously described (37). Cytotoxicity against HIV-expressing CD4 lymphocytes was studied in parallel. Similar
to the results reported above, effector cells from HIV-infected children were unable to lyse HIV-expressing CD4 lymphocytes (Table 3). Addition of HIV-seropositive serum
from an adult to the effector cells of HIV-infected children did not
significantly increase lysis of HIV-expressing CD4 cells. Effector
cells of age-matched HIV-seronegative children were able to kill these
targets to a significantly greater extent in the presence of serum from
the same HIV-seropositive adult than in the presence of serum from a
seronegative adult (P < 0.05).
In contrast to results with HIV-expressing CD4 lymphocytes, effector
cells from HIV-infected children lysed HSV-infected FS4 target cells to
a significantly greater extent in the presence of an HSV-seropositive
serum sample than in the presence of an HSV-seronegative serum sample
(P < 0.05). In addition, effector cells from
HIV-infected children were able to lyse HSV-infected targets to the
same extent as did effector cells from HIV-seronegative children. These
results suggest that NK cells from HIV-infected children are capable of
mediating ADCC against target cells infected with a virus other than HIV.
PBMC from HIV-infected adults lysed HIV-expressing lymphocytes
equally in the presence and in the absence of HIV-seropositive serum (P > 0.05). Specific cytotoxicity was mediated
by PBMC of HIV-seronegative controls when an HIV-seropositive serum
sample was added to assay mixtures (P < 0.05). These
results were consistent with previous reports that in vitro lysis of
HIV-expressing CD4 lymphocytes is mediated by HIV patients' effector
cells armed in vivo with cytophilic antibodies (57, 62).
Similar to the observations with children, PBMC from HIV-infected
adults and those from HIV-seronegative adults were equally effective in
lysing HSV-infected targets in the presence of anti-HSV
antibody-containing sera (P > 0.05).
To further characterize the functional defect in HIV-infected children,
we tested the ability of their PBMC to lyse HUT78 cells chronically
infected with the IIIB strain of HIV and uninfected HUT78 cells. PBMC
from HIV-infected children and those from uninfected children gave
similar results with respect to the ability to lyse uninfected or
infected HUT78 cell targets (Table 4).
These observations, along with the results for ADCC against
HSV-infected targets, indicate that antibody-dependent and -independent
NK cell-mediated cytotoxicity is generally intact in perinatally
HIV-infected children.
To determine whether deficient cytotoxicity against HIV-expressing CD4
cells in HIV-infected children was due to deficient production or to
effector cell binding of ADCC-mediating antibodies, assays were carried
out by addition of serum from an HIV-seropositive adult throughout the
4-h incubation of the ADCC assay. The presence of seropositive serum
was considered to increase the magnitude of cytotoxicity if the mean
lysis was higher than the mean cytotoxicity plus 2 standard deviations
in the presence of HIV-seronegative serum. For 7 of the 15 HIV-infected
children tested, addition of HIV-seropositive serum from an adult had a
positive effect on the magnitude of cytotoxicity (Table
5). The presence of adult HIV-seropositive serum also resulted in increased lysis mediated by
effector cells of uninfected children.
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TABLE 5.
Lysis of IIIB-coated CD4 lymphocytes mediated by PBMC of
HIV-infected children in the presence of adult serum
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|
In further studies, we added sera from children with perinatal HIV
infection to ADCC assays in which PBMC from HIV-seropositive adults
were tested for their ability to lyse HIV-expressing CD4 lymphocytes.
As shown in Fig. 3a, the addition of
individual sera from seven pediatric HIV patients in seven experiments
each time inhibited cytotoxicity mediated by adult PBMC (P < 0.05). In contrast, the presence of sera from uninfected
children had no effect on the magnitude of ADCC mediated by PBMC from
HIV-infected adults (Fig. 3b).
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FIG. 3.
Effects of sera from HIV-seropositive (a) and
HIV-seronegative (b) children on cytotoxicity against IIIB-coated CD4
lymphocytes mediated by PBMC of HIV-infected adults () at early
stages of disease and by PBMC of an HIV-seronegative adult ().
|
|
| |
DISCUSSION |
In HIV-infected individuals, humoral and cellular immune
mechanisms may act to reduce the quantity of virus in cells and
tissues. Studies on the role of cell-mediated cytotoxicity in
HIV-infected adults have yielded contradictory results (5, 13, 14,
20, 21, 28, 38, 42, 46, 53, 58). Several recent publications have
addressed the correlation between clinical stages of infection and
various types of cytotoxic activity detected in vitro, including cytotoxic T-lymphocyte-mediated cytotoxicity, NK cell-mediated cytotoxicity, and ADCC (1, 20, 26-28, 38, 58, 61). In some
studies, reduced ADCC (1, 45, 58) and NK cell-mediated cytotoxicity correlated with disease progression (20, 52, 58,
61), whereas in another study increased ADCC activity was
detected in patients with AIDS compared to those at earlier stages of
infection (38). Some investigators did not find any correlation between cytotoxic activity and clinical stage of HIV infection (26-28). Others described a disparity between NK
cell-mediated cytotoxicity, which was observed to decline around the
time of first symptoms, and ADCC activity, which remained constant
throughout the course of disease (19). In all these ADCC
studies with PBMC from adults, HIV immune globulin was added to the
assay and HIV-infected T-cell lines were used as targets. Different
results among these studies may be explained by the different virus
strains, target cell lines, or test conditions employed in the
cytotoxicity assays. Even subclones of the same cell line may vary
significantly in their susceptibility to ADCC (48). For this
reason, we decided to use effector cells, armed in vivo with
ADCC-mediating antibodies, to measure cytotoxicity against target cells
which appear to be one of the major reservoirs for HIV in infected
humans, namely, CD4 lymphocytes.
We showed that PBMC of vertically HIV-infected children were impaired
in their ability to lyse HIV-expressing CD4 lymphocytes. In contrast,
PBMC of HIV-infected adults lysed such target cells, and the magnitude
of lysis correlated with absolute numbers of circulating CD4
lymphocytes. This cytotoxicity was shown to be mediated by NK cells
armed in vivo with cytophilic HIV antibodies, confirming previous
reports (57, 62). We found normal percentages of
CD16+ NK cells in HIV-infected children, confirming
previously published data (34).
The inability of PBMC from children with vertical HIV infection to
mediate such ADCC against CD4 lymphocytes expressing HIV antigens was a
specific and functional, rather than a quantitative, deficiency:
effector cells from the same children lysed HSV-infected fibroblasts in
ADCC assays and killed HIV-infected HUT78 cells and K562 cells in NK
cell-mediated cytotoxicity assays to the same extent as did PBMC from
HIV-seronegative children.
Attempts to induce ADCC against HIV-expressing CD4 lymphocytes, by
using effector cells of HIV-infected children and serum of an
HIV-seropositive adult, gave variable results. PBMC of about one-half
of the children showed ADCC-mediated lysis when antibody-containing serum of an HIV-infected adult was added directly to the assays. Furthermore, sera from HIV-infected children interfered with the ability of PBMC from infected adults to mediate ADCC against CD4 lymphocytes. Thus, factors capable of blocking ADCC against
HIV-expressing CD4 cells may be present in sera of perinatally
HIV-infected children.
NK cells are present in human fetal liver mononuclear cells by the 8th
week of gestation (40), mediate cytotoxicity as early as the
9th gestational week (59), and are functionally mature by
the 32nd week (47). However, decreased NK cell activity
against HIV-expressing targets, as reported for premature (<35 weeks
of gestational age) neonates (35), may be due to antenatal
glucosteroids, fetal stress, or other critical care issues. Other
investigators of pediatric cell-mediated immune responses reported
normal NK cell activity and decreased ADCC activity against
HIV-expressing target cells in HIV-seropositive neonates (17,
41). Interestingly, HIV-1 gag-cytotoxic T-lymphocyte responses
were reported to be also deficient in vertically HIV-infected children
(30). Our data indicate that vertical HIV infection does not
interfere with maturation of functional NK cells. PBMC of HIV-infected
children mediated cytotoxicity against IIIB-infected HUT78 cells in NK cell assays as well as against HSV-infected fibroblasts in ADCC assays.
The levels of cytotoxicity observed were comparable to those obtained
with PBMC from age-matched healthy peers.
It has been previously shown that decreased NK cell-mediated lysis in
AIDS patients could be restored in vitro by addition of cytokines to
cytotoxicity assays (1, 24, 25, 44). Bonavida et al.
(6) reported that IL-2 triggered release of NK cell
cytotoxic factors from PBMC of HIV-infected adults. Ahmad et al.
(1) showed in vitro that the addition of IL-2 or gamma interferon enhanced ADCC activity significantly in PBMC from AIDS patients (CD4 counts of <200), which had without cytokine addition significantly lower target lysing ability than those from patients with
CD counts of >400. Positive in vitro effects of other cytokines, such
as IL-12 and IL-15, on ADCC-mediated lysis in pediatric HIV patients
have been reported elsewhere (25). Other cytokines are still
under investigation. However, addition of IL-2 to effector cells of
HIV-infected children did not enhance their ability to mediate lysis of
HIV-expressing CD4 cells (data not shown).
ADCC against HIV-expressing CD4 lymphocytes is mediated by NK cells of
HIV-infected adults, linked in vivo to antibodies of the subclass IgG1
(27). Such antibodies are directed against the viral
envelope glycoproteins gp120 and gp41 (12, 15, 22, 31, 33, 51, 55,
56, 60, 62, 64) and are distinct from virus-neutralizing
antibodies (7, 32, 50). Both ADCC-mediating antibodies and
neutralizing antibodies were shown to be present in sera of infants
born to HIV-infected mothers (19, 29). The presence of such
antibodies, most likely of maternal origin, correlated with a better
clinical outcome in one study (32) but had no clinical
significance in another investigation (18). However, both
reports confirm that the presence of ADCC antibodies failed to prevent
vertical transmission of HIV infection. Hypergammaglobulinemia with
high levels of IgG1 and IgG3 has been detected in perinatally infected
children, but such children frequently have functional hypoglobulinemia
and reduced defenses against bacterial opportunistic infections
(43). A similar functional inability of antibodies to
mediate ADCC against HIV-expressing CD4 lymphocytes might explain the
results observed in the present study.
Blocking factors in sera of HIV-infected children might inhibit the
arming of effector cells or the contact between effector and target
cells. Immune complexes, known to be present more frequently and in
higher concentrations in HIV-infected children (10, 27) than
in adults (36), might act as such blocking factors.
Differences in non-HIV-specific immunoglobulin, HIV-specific
noncytophilic antibody, or cytokine profiles between sera of pediatric
patients and sera of adult patients may also be accountable for these
inhibitive effects.
In summary, our results are similar to several reports about deficient
ADCC activity against HIV-expressing target cells in pediatric
HIV-infected populations (17, 35, 54) and noncompromised NK
cell activity (17, 35). In addition, we demonstrate that the
deficit in ADCC activity in HIV-infected children is not due to
defective effector cells, appears to be specific to HIV-infected ADCC
target cells, and may be caused by undefined serum blocking factors.
This deficiency may be a contributing factor to the rapid disease
progression often observed with this patient population.
| |
ACKNOWLEDGMENTS |
We thank Susan Plaeger and Kenneth Ugen for their extremely
valuable help and advice; Barbara Frank for graphic design; and Tracy
Gamble, Evan Crawford, and Sihuor Peak for the preparation of the
manuscript. Most importantly, we appreciate the patients' willingness
to participate in our study and, above all, our pediatric patients'
valuable blood donations, which made this study possible.
This project was supported by a grant of the Deutsche
Forschungsgemeinschaft, the Pediatric AIDS Foundation, and the
University of Pennsylvania Research Foundation. U. Ziegner was a
scholar of the Pediatric AIDS Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: UCLA Children's
Hospital, Division of Allergy and Immunology, MDCC, Rm. 22-387, Los Angeles, CA 90095. Phone: (310) 825-6481. Fax: (310) 206-5843. E-mail: uziegner{at}mednet.ucla.edu.
| |
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