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Basic Science

Development of T-Cell Lines Expressing Functional HIV-1 Envelope Glycoproteins for Evaluation of Immune Responses in HIV-infected Individuals

Keler, Tibor; Li, Hongbo*; Cloyd, Miles W.*; Vitale, Laura A.; Deo, Yashwant M.

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Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology: October 1, 1996 - Volume 13 - Issue 2 - p 117-126
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Abstract

The role of antibody-dependent cellular cytotoxicity (ADCC) in human immunodeficiency virus (HIV) immunosurveillance remains poorly understood and the subject of controversy. HIV-seropositive individuals at different stages of disease have variable levels of circulating antibodies that mediate ADCC in vitro (reviewed by Brenner et al. [1]). Although these antibodies mediate ADCC in vitro, their role in protection from HIV-mediated disease is not clear. Some studies have documented that a decrease in the level of antibody titers that mediate in vitro ADCC correlate with advanced disease state (2-4). In addition, the presence of antibodies functional in in vitro ADCC and viral neutralization was correlated with better clinical status and delayed disease progression in HIV-infected infants (5,6). However, other studies were unable to demonstrate a link between ADCC antibody titers and clinical stage or clinical outcome (7-9). Investigations of the cellular aspect of ADCC in HIV-infected persons have primarily focused on natural killer cell-mediated ADCC and have established an impairment in this effector cell population in HIV-infected persons (4,10-12). However, the nature of this defect and its relationship to disease progression is unresolved.

The predominant target antigen for ADCC expressed by HIV-infected cells is env(13). The HIV env is processed from noncovalently linked oligomers of gp160 precursor into oligomers containing gp41 and gp120 env subunits (reviewed by Freed and Martin [14]). The gp41/120 glycoproteins are anchored in the plasma membrane of infected cells by the gp41 transmembrane domain, exposing a part of gp41 and the associated gp120. HIV env functions in directing the cellular tropism of the virus and modulating immune responses. The humoral and cellular immune responses elicited by HIV env appear to play a significant role in HIV pathogenesis and therefore have been the focus of vaccine development. The importance of HIV env oligomer formation to eliciting functional immune responses has become evident and has led to a special emphasis on conformational epitopes present in the gp41/gp120 complex (15-17).

Current methods for studying ADCC of HIV-infected cells are limited. The major difficulties associated with in vitro ADCC models include heterogeneity of antigen expression on target cells, interference from non-ADCC-mediated cell death resulting from the cytopathic effects of virus infection, non-native presentation of HIV antigens, and issues concerning practicality and safety when assays need to be conducted routinely. A variety of target cells have been used to study ADCC-mediating anti-HIV antibodies, including chronically infected cell lines (18), target cell lines infected with vaccinia virus vectors encoding HIV proteins (8), target cells coated with virus, recombinant gp120 or peptides (12,19,20), and gp120 or gp160 transfected cell lines (3,4). Each system offers advantages for different applications. The transfected cell lines that express stable levels of antigen are of particular value when large numbers of samples are to be evaluated in a reproducible and safe manner. Transfected target cells can be used to evaluate ADCC activity toward specific antigens without interference from other viral or irrelevant proteins that may hinder interpretation of results. Most important, env antigens expressed on transfected cells that are appropriately processed can react with antibodies that are specific for conformational epitopes. Although T-cell lines transfected with gp160 have been described for use as targets for ADCC (3,21), efficient processing of gp160 to yield functional surface gp120 and gp41 has not been demonstrated. Furthermore, since CD4 expression is not significantly down-regulated on the surface of these gp160 transfected cells, gp120 is complexed with CD4 on the surface of the cells (21). Such interactions are likely to affect the binding ability of antibodies specific for the CD4 binding site of gp120 and antibodies that recognize certain conformational epitopes in gp120 and gp41.

The goal of this study was to develop and characterize HIV env-expressing cell lines, which retain functional and antigenic properties of the native proteins. This report describes the characterization of three transfected cell lines expressing HIVpm213env derived from CEM cells, a human T-lymphoid cell line. HIV-1pm213 was isolated in the laboratory of R. Gallo and has been characterized for reactivity with sera from HIV-1 infected patients (22). This strain belongs to the B clade, predominant in North America and Europe, and has antigenic determinants that are recognized by sera from HIV-1-infected individuals in the early phase of infection in the absence of seroconversion (23). Therefore, this strain was thought to be ideal for expression of common antigenic determinants of HIV-1 env. The env-expressing cell lines demonstrated stable high-level expression of surface gp41/gp120, which was correlated with a specific reduction of surface CD4. The env-expressing cell lines mediated syncytia formation with CD4-positive cells, were specifically recognized by immunoglobulins from sera of HIV-infected individuals or monoclonal antibodies specific for gp120 and gp41, and were selectively lysed by ADCC with the HIV-seropositive sera. These model cell lines may be valuable for functional analysis of cellular and humoral ADCC activity at various stages of HIV infection. In addition, the cell lines provide a practical method for evaluating ADCC titers in response to vaccines and the ADCC potential of anti-HIV env antibodies for therapeutic use.

MATERIALS AND METHODS

Antibodies and Reagents

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HeLaT4 and HeLaT8 cells from Dr. Richard Axel (24) and rabbit anti-CD4 serum from Dr. R. Sweet, SmithKline Beecham Pharmaceuticals (25). OKT3- and OKT4-producing hybridomas were purchased from American Type Culture Collection (Rockville, MD, U.S.A.). Murine anti-gp120 antibody gpIII2-3 was purified from hybridoma culture supernatant by protein A affinity chromatography. The human anti-gp41 antibody 126-D was generously provided by Dr. Susan Zolla-Pazner (New York University, New York City). Serum samples from HIV-infected persons were kindly provided by Dr. B. Vandercam (Université Catholique de Louvain, Brussels, Belgium) and Dr. J. L. Pasquali (H.U.S. Hôpital Civil, Strasbourg, France).

Generation of HIV-1 Envelope-expressing Cell Lines

CEM cells were transfected by electroporation with pLNSX plasmid (26) containing a SalI-XhoI insert spanning the env sequences from HIV-1213(27). Clones resistant to Geneticin (Gibco BRL, Grand Island, NY, U.S.A.) were isolated and further propagated in selection medium. Three clones that stained positive for gp120 were chosen for further characterization and designated CEM-213env1, CEM-213env4, CEM-213env7. Cells were maintained in Iscove's modified Dulbecco medium (IMDM) supplemented with 10% fetal calf serum (FCS) and 0.4 mg/ml Geneticin.

Flow Cytometry

CEM and CEM-213env cell lines were washed with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (PBA) and added to 96-well plates at 3 × 105/well. The plate was put on ice, and antibodies/antiserum diluted in PBA or hybridoma supernatant was added to wells in an equal volume. Where appropriate, an irrelevant antibody of similar species and isotype was used to determine nonspecific binding. Binding was allowed to continue for 1 h on ice, followed by three washes with PBA. Fluorescein isothyocyanate (FITC)- or phycoerytherin (PE)- conjugated species-specific antibodies were added at appropriate dilutions in 50 μl and incubated on ice for 1 h. The second-step reagents used were goat antimurine immunoglobulin G (IgG) (-FITC 1:40, -PE 1:50; Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.), goat antihuman IgG (-FITC 1:50, -PE 1:50; Jackson IRL), donkey antirabbit IgG-FITC (1:30; Jackson IRL). Following incubation with FITC or PE probes, the cells were washed three times with PBA and resuspended in 400 μl of 1% paraformaldehyde in PBS. Samples were analyzed on Becton Dickinson FACSan.

Western Blot Analysis

Cell lysates were prepared from cell cultures by resuspending 2 × 107 cells in 2 ml of PBS containing 0.5% Triton-X 100 and aprotinin, 0.2 TIU/ml. Lysates were prepared on ice with occasional vortexing. After 60 min, insoluble cell debris was removed by centrifugation at 15,000 g for 15 min. Total protein concentrations were determined with the BioRad protein assay (BioRad, Hercules, CA, U.S.A.). Lysates were diluted in nonreducing sample-loading buffer containing 2% sodium dodecyl sulfate (SDS) or with reducing sample-loading buffer containing 10% SDS and 2-β-mercaptoethanol. The lysates were loaded at 30 μg total protein per well on preformed 4-15% gradient gels (BioRad). After electrophoresis, proteins were transferred to nitrocellulose, and the blots were processed with the ECL Western Blotting Kit (Amersham, Arlington Heights, IL, U.S.A.). Antibody incubations were as follows: gpIII2-3, 3 μ/ml, and horseradish peroxidase-conjugated antimurine IgG at a 1:10,000 v/v dilution.

ADCC

Mononuclear effector cells were purified from heparinized whole human blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Piscataway, NJ, U.S.A.). These effector cells were added to 96-well U-bottom microtiter plates at 3 × 105 cells/well in IMDM supplemented with 10% FCS. Target cells (control CEM and env-expressing cell lines) were labeled for 1 h with 0.1 mCi of 51Cr (Amersham) and added to effector cells at 1 × 104 cells/well in IMDM supplemented with 10% FCS. Anti-HIV antibody or sera were diluted in the same media and added at various concentrations. Plates were incubated 14-16 h at 37°C in a humidified incubator with 5% carbon dioxide. Samples of supernatant were harvested for determination of radioactivity. Maximum lysis was determined from target cells incubated in 2% SDS. The spontaneous release (target leak) was ≈20% of the maximum release (detergent lysis). All samples were tested in triplicate and evaluated by the formula% lysis = (experimental CPM - target leak CPM/maximum lysis CPM - target leak CPM) × 100%. Specific lysis was calculated by subtracting the percentage lysis without antibody from the percentage lysis with antibody.

Syncytia Assay

Cultures of HeLaT4 and HeLaT8 cells (obtained through the AIDS Research and Reference Reagent Program) were harvested by trypsinization. HeLa cells (5 × 104) were combined with an equal number of CEM, CEM-213env1, env4, or env7 cell lines in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide. Adherent cells were fixed and stained with Diff-Quick (Sigma Chemical Co., St. Louis, MO, U.S.A.) before microscopic observation.

RESULTS

Transfection and Selection of gp160-expressing Cell Clones

The env from HIVpm213 was chosen for cloning and expression in the CEM lymphoid T-cell line since previous studies had demonstrated that this isolate contains broadly conserved epitopes (22). HIVpm213 DNA, including the entire env sequence, part of vpr, and the first exon of tat, rev, and vpu, was blunt-end ligated into the StuI cloning site of the vector pLNSX (Fig. 1). In this vector the env gene is driven by the SV-40 promoter, and the neomycin resistance gene is driven by the long terminal repeat. The vector was transfected into CEM cells by electroporation, and clones were selected in media containing G-418 antibiotic. Positive cultures were identified by a live cell immunofluorescence technique using sera from HIV-seropositive donors. Three positive clones, CEM-213env1, CEM-213env4, and CEM-213env7 were selected, expanded, and further characterized.

Cell Surface Expression of gp41 and gp120

The stability and amount of env expression was monitored by flow cytometry with monoclonal antibodies specific for gp120 and gp41. Sample histograms of control and env-expressing cell lines are shown in Fig. 2. The env-expressing clones routinely stained >95% positive for gp120 and 50-90% positive for gp41. The CEM-213env1 and -env4 cell lines maintained the surface expression of gp41 and gp120 in continuous culture for at least 24 passages (6 months) when cultured in selection media (Table 1). In the absence of selective pressure, the levels of env expression decreased; however, after 10 passages >95% of the cells remained positive for gp120 with a mean fluorescence intensity eight- to 12-fold greater than the control cells. In order to evaluate the ability of natural antibody responses to recognize the transfected env, CEM-213env1 and CEM cells were reacted with serum samples from nine HIV-infected individuals. All nine patients' samples specifically reacted with CEM-213env1 cells with moderate to high binding (Table 2).

Processing of gp160

Further characterization of glycoprotein expression and processing was evaluated by Western blots. Using the anti-gp120 monoclonal antibody, gpIII2-3, cell lysates of CEM-213env1, -env4, and -env7 were analyzed for gp120/gp160 expression under reducing and nonreducing conditions. Figure 3 shows that under reducing gel conditions, the CEM-env cell lysates contained predominantly gp120 (estimated mol. wt. = 125 kDa). The absence of significant staining of gp160 suggests that the majority of gp160 is cleaved into gp120 and gp41. It is important to note that nonreducing gels demonstrated the presence of high-molecular-weight aggregates, >200 kd, suggesting that oligomers were present in the cell lysates.

Syncytia Formation

In order to address the functional activities of the transfected glycoproteins their ability to form syncytia with CD4-expressing cells was examined. When CEM-213env1 cells were co-cultured overnight with HeLaT4 cells (CD4+), numerous large syncytia were observed, in contrast to control cultures with CEM cells (Fig. 4). Similar numbers and sizes of syncytia were apparent in HeLaT4 cocultures with CEM-213env-4 or env-7 cells (data not shown). No syncytia were detectable with the control CEM cells or when the env-expressing cells were co-cultured with HeLaT8 cells (CD4-, CD8+) (data not shown). The number of nuclei in the fused cells varied greatly and ranged from 2 to 16. These results indicate that the HIV env expressed on the transfected cell lines was appropriately processed and biologically functional.

CD4 Modulation

The lack of visible syncytia in the env-expressing cultures, in the absence of HeLaT4 cells, suggested that the surface CD4 levels on these cells may be down-regulated. To address this possibility, the level of surface CD4 was analyzed by flow cytometry with anti-CD4 rabbit sera. Table 3 summarizes these results. The surface CD4 expression was acutely down-regulated on all three cell lines compared with control. Only 20-30% of the transfected cells had detectable surface CD4, with a mean fluorescence ≈10% of control cells. Down-modulation was specific to the CD4 receptor since the surface levels of the CD3 receptor were not lowered or, in the case of CEM-213env1, were consistently upregulated. The regulation of surface CD4 expression suggests that transfected HIV env was processed and was functional in a manner similar to env expressed in a virally infected cell.

Specific Lysis of CEM-213env by ADCC

The env-expressing cells were evaluated as targets for ADCC. To demonstrate target specificity, untransfected and CEM-213env cell lines were labeled with 51Cr and combined with mononuclear cells from normal donors and anti-gp120 monoclonal antibody (Fig. 5). The env-expressing cell lines, and not controls, were lysed in an antibody-dependent manner. The CEM-213env7 cells were the least sensitive to ADCC, which is consistent with their relatively lower gp120 expression (see Fig. 2). The low level of ADCC with the monoclonal antibody gpIII2-3 may be related to its isotype, murine IgG1, which may restrict the types of effector cells engaged for ADCC (28).

Heat-inactivated sera from HIV-infected persons, in nine or nine cases, mediated efficient ADCC of CEM-213env cells (Fig. 6). The ADCC of CEM-213 env1 cells ranged from 28 to 45% at a 1:100 dilution of the sera from HIV-infected individuals. Under the same conditions, the CEM-213env4 and env7 cells were killed less efficiently, with a range of 8-38%. The control CEM ADCC was negative for most sera and ranged from - 12% to 7%. The negative ADCC values may be due to inhibition of spontaneous lysis by the patient's sera. ADCC of env-expressing cells ranged from 5 to 39% at a 1:500 dilution of sera. Interestingly, two of the samples from HIV-infected persons (no. 1 and no. 7) appeared to mediate low-level ADCC of the CEM cells that did not express HIV env. One of these samples (no. 1) also demonstrated some binding to CEM cells (Table 2). The ADCC experiments show that the HIV env-transfected cell lines express antigenic determinants that are recognized by monoclonal and polyclonal antibodies leading to target cell destruction.

DISCUSSION

The human T-cell line, CEM, was transfected with the expression vector pLNSX containing the complete sequences from HIV-1pm213env. The transfected clones, CEM-213env1, env4, and env7, maintained high-level expression of surface gp120 and gp41, which made them suitable targets for ADCC with anti-gp120 monoclonal antibody and sera from HIV-infected individuals. Furthermore, the expression of env proteins resulted in near elimination of surface CD4 and the ability to form syncytia with CD4-expressing HeLa cells. To our knowledge, this is the first report describing stable cell lines with high-level surface expression of processed and functional HIV gp41 and gp120 envelope glycoproteins.

Flow cytometric analysis and Western blots confirmed the expression and processing of env. Significantly higher surface expression was observed with the anti-gp120 monoclonal antibody than with anti-gp41 monoclonal antibody. This difference may be due at least in part, to the greater accessibility of the gp120 epitope (gpIII2-3 is specific for the V3 loop of gp120) rather than to a quantitative difference in their expression. Analysis of the relative levels of gp41 and gp120 by solid phase immunoassay (enzyme-linked immunosorbent assay, or ELISA) of cell lysates did not demonstrate significant differences between reactivity with antibodies specific for the individual glycoproteins (data not shown). Despite the high level of env surface expression, soluble gp120 in the culture supernatants was undetectable by ELISA (data not shown). The lack of gp120 shedding is advantageous for ADCC assays in which the soluble gp120 may interfere through the formation of immune complexes that bind to Fc receptors. Although both gp41 and gp120 were detected on the surface of the transfected cells, flow cytometry analysis could not differentiate between unprocessed gp160 and the cleaved products. Therefore, immunoblot analysis of lysates from the transfected cells probed with anti-V3 loop monoclonal antibody gpIII2-3 was used to demonstrate that nearly all of the gp160 was processed to gp120 and presumably to gp41. This monoclonal antibody reacts with both gp120 and gp160 in HIV-1pm213-infected cells (data not shown). Since proteolytic cleavage of gp160 is believed to occur after and be dependent upon oligomer formation (14,29), it is likely that env in the transfected cell lines is processed in native oligomeric forms. In fact, electrophoretic separation of the cell lysate under nonreducing conditions showed that a significant amount of the glycoproteins was present in high-molecular-weight aggregates. Since viral membrane glycoprotein oligomers have been shown to be resistant to detergent solubilization, the aggregates found in the CEM-213env lysates likely represent naturally formed oligomers of gp41 and gp120, which are fundamental to many env-related functions, including cell fusion (14,29,30).

The processing and expression of functional env was further evidenced by the ability to form syncytia with CD4-positive cells. The env-transfected cells formed abundant and large syncytia upon co-culture with CD4+ HeLaT4 cells. Since syncytia formation requires cleavage of gp160 into its subunits and proper surface expression (29,30), these data further support processing of gp160 in the env-expressing cells. Syncytia were not evident in CEM-213env cultures, even though the CEM cells from which they were derived express high levels of CD4. This was apparently due to a nearly complete loss of surface CD4 expression. Approximately 70-80% of the env-expressing cells had no detectable surface CD4, and the remaining cells had levels equivalent to about one tenth of the level on control CEM cells. This low-level surface expression of CD4 on some of the env-expressing cells may be due to binding of soluble CD4 released from dead cells to gp120. The ability to down-regulate surface CD4 expression has been attributed to several HIV proteins, including env(31,32). However, there are contradictory reports on the ability of env expression to lead to significant reduction in surface CD4 in stably transfected cell lines (3,21,32). This study clearly shows the effect of env expression on surface CD4 expression. The lack of CD4 modulation reported by Ahmad et al. (4) may reflect differences in the env sequences or possibly a quantitative difference in the level of env expression. High levels of env expression may be required before a reduction in surface CD4 can be detected. The env alone is most likely responsible for CD4 modulation in the CEM-213env cells, since the vpu, which has been shown to contribute to CD4 down-regulation (26,31), encoded by of HIVpm213 contains a mutation in the initiation codon preventing translation of vpu (unpublished results). Furthermore, no env expression or modulation of surface CD4 was observed in CEM cells transfected with the vector alone (data not shown).

Recombinant env was recognized by HIV sera from nine of nine infected individuals, substantiating the presence of conserved antigenic determinants. Sera from the same patients mediated ADCC of the env-expressing cell lines, suggesting that they will provide a suitable model for studying functional antibodies in relation to HIV pathogenesis and therapy. To define the ADCC-mediating antibody titers in HIV-infected persons and their relationship to pathogenesis, it is important that the target cells used for such assays react with conformationally dependent antibodies in addition to antibodies that recognize linear epitopes. The presence of conformationally dependent antibodies are thought to be particularly important in neutralization of primary isolates of HIV (15-17) and may have similar relevance in ADCC. Interestingly, in a study of a population at high risk for HIV seroconversion, ADCC-mediating antibodies were found to precede neutralizing antibodies and seroconversion, as measured by solid-phase immunoassays (33). Furthermore, samples from HIV-infected individuals that were seronegative by standard tests (ELISA and Western blot) were found to contain antibodies that reacted specifically with cells infected with HIV by a live cell immunofluorescence assay (24). These studies suggest that ADCC may be particularly important in controlling the early spread of infection and that antibodies in the primary humoral response are targeted to conformational epitopes.

The env-expressing cells described in this report express processed and functional env with conserved antigenic determinants. Therefore, these cells should be ideal for studying ADCC antibody titers in HIV-infected patients, particularly in the early phase of infection, when antibodies to conformational epitopes may be prevalent. In addition to the humoral aspects of anti-HIV ADCC, it is expected that these cells will be useful for evaluating the effector cell functions in HIV-infected individuals. Interestingly, some of the HIV-seropositive samples (no. 1 and no. 3, Fig. 6) mediated similar levels of ADCC with either the high-expressing CEM-213env-1 or the lower-expressing CEM-213env-7 cells, suggesting the presence of unique antigenic determinants on the individual cell lines. Expression of additional env genes from other HIV strains will be useful for studying the cross-reactivity of ADCC antibody titers with sera from patients infected with HIV from various clades. In addition to studying the role of ADCC in HIV infection, the CEM-213env cells may serve as useful targets for evaluating the efficacy of candidate env-based vaccines to elicit ADCC-mediating antibodies and for developing antibodies with therapeutic potential.

Acknowledgment: The authors gratefully thank Ellen Clinebell for her excellent clerical and editorial contributions. Hungbo Li was supported by a McLaughlin Research postdoctoral fellowship.

F1-2
FIG. 1:
. Schematic representation of HIVpm213 env expression vector.
F2-2
FIG. 2:
. Surface expression of gp41 and gp120 on transfected cell lines. Representative histograms of each env-transfected cell line are shown after staining with irrelevant isotype-matched monoclonal antibody (shaded curve), anti-gp41 monoclonal antibody 126-D (dotted line), and anti-gp120 monoclonal antibody gpIII2-3 (solid line) and compared with control CEM cells. The y axis represents relative cell numbers. Cells were stained with 20 μg/ml primary antibody followed by 1:40 dilution of PE-conjugated species-specific second antibody. Reactions were done on ice for 1 h each before fixing cells in 1% paraformaldehyde.
T1-2
T2-2
F3-2
FIG. 3:
. Western blot analysis of gp120 in CEM cell lysates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 4-15% polyacrylamide gradient gels under nonreducing (A) and reducing (B) conditions. The gels were transferred to nitrocellulose and probed with the murine monoclonal antibody gpIII2-3 (anti-gp120). The Western blots were developed by chemiluminescence using a horseradish peroxidase-conjugated antimouse second antibody. Lane 1, CEM; lane 2, CEM-213env1; lane 3, CEM-213env4; lane 4, CEM-213env7. Each lane contained 30 μg of total protein from cell lysates.
F4-2
FIG. 4:
. Syncytia formation with CEM-213env1 cells and HeLaT4 cells. CEM controls (left) or CEM-213env1 cells (right) were co-cultured overnight with HeLaT4 cells in 24-well tissue culture plates at a 1:1 ratio. After incubation, nonadherent cells were removed with a PBS wash, and remaining cells were fixed and stained with a modified Wright stain and examined by phase-contrast microscopy. Magnification ×200.
T3-2
F5-2
FIG. 5:
. ADCC of env-transfected cell lines by mononuclear cells mediated by anti-gp120 monoclonal antibody. Control CEM cells (open circles) and HIV env-expressing clones CEM-213env1 (solid circles), env-4 (solid triangles) and env-7 (solid squares) were labeled with 51Cr and then incubated with normal mononuclear cells and various concentrations of the antibody gpIII2-3. The effector-to-target ratio was 30:1. After a 16-h incubation, the percentage ADCC was calculated from triplicate wells and presented as the mean ± SD.
F6-2
FIG. 6:
. ADCC of env-transfected cell lines by mononuclear cells mediated by sera of HIV-infected individuals. Control CEM cells and env-expressing clones were labeled with 51Cr and incubated with mononuclear cells in the presence of sera (1% final concentration) collected from HIV-infected persons. The effector-to-target ratio was 30:1. After a 16-h incubation, the percentage ADCC was calculated from triplicate wells and presented as the mean ± SD. CEM controls that resulted in a negative percentage ADCC were plotted as 0% ADCC. The actual values ranged from -2 to -12%, with all SD <5%. The percentage lysis in the presence of normal human serum was CEM = 11%, CEM-213env1 = 34%, CEM-213env4 = 35%, and CEM-213env7 = 54%.

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Keywords:

HIV envelope; CD4 modulation; Syncytia; Antibody-dependent cellular cytotoxicity

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