MATERIALS AND METHODS
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
), 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.
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 FcγIII receptor (CD16) on NK cells, as previously described (3
) 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
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%. Percent51Cr 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.
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.
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
). 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
). In some studies, reduced ADCC (1
) and NK cell-mediated cytotoxicity correlated with disease progression (20
), 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
). 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
). 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
). 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
) and are distinct from virus-neutralizing antibodies (7
). Both ADCC-mediating antibodies and neutralizing antibodies were shown to be present in sera of infants born to HIV-infected mothers (19
). 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
) 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
) and noncompromised NK cell activity (17
). 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.