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A Semiautomated Fluorescence-Based Cell-to-Cell Fusion Assay for gp120–gp41 and CD4 Expressing Cells
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
240, 49–57 (1998)
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A Semiautomated Fluorescence-Based Cell-to-Cell Fusion Assay for gp120–gp41 and CD4 Expressing Cells P. Scott Pine,*,1 James L. Weaver,* Tamas Oravecz,† Marina Pall,† Michael Ussery,‡ and Adorjan Aszalos* *Division of Applied Pharmacology Research, CDER, Food and Drug Administration, Laurel, Maryland 20708; †Division of Hematologic Products, CBER, Food and Drug Administration, Bethesda, Maryland 20708; and ‡Antiviral Research Laboratory, CDER, Food and Drug Administration, Rockville, Maryland 20708
ing cells, has been used as an endpoint for measuring various inhibitors of HIV infection [1, 8]. Transfected cell lines, which express complementary components of the fusion reaction, can be used as models of cell-tocell fusion and avoid some of the problems associated with using live viruses [9, 10]. We present a quantitative assay based upon fluorescence image analysis of cell-to-cell fusion using CD4/ cells, either Sup-T1 cells or CD4/-enriched human peripheral blood lymphocytes (PBL), and env/ TF228.1.16 cells, BJAB cells which stably express the gp120–gp41 complex. This assay is performed in a multiwell plate and can be completed in less than a day. We show that changes in the two-dimensional distribution of the fluorescent indicator, calcein, can be used to measure cell-to-cell fusion and syncytium formation. The method was shown to yield quantitative results for prevention of syncytium formation by evaluating several compounds known to act at different sites in the HIV infection process.
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4/ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120–gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4/ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cellto-cell fusion. q 1998 Academic Press
MATERIALS AND METHODS Reagents. Aurin tricarboxylic acid (ATA) and cytochalasin D were from Sigma (St. Louis, MO). The peptide T20 was obtained from Dr. Dennis M. Lambert (Trimeris Inc., Research Triangle Park, NC) by material transfer agreement. T20 is a 36-amino-acid synthetic peptide derived from a domain within gp41. The monoclonal antibody (mAb) anti-Leu3a was from Becton Dickinson Immunocytometry (San Jose, CA); OKT4 and OKT4E were from Ortho Diagnostic Systems (Raritan, NJ). Cells and culture conditions. TF228.1.16 cells and BJAB cells were obtained from Dr. Z. Jonak at SmithKline Beecham Pharmaceuticals (by material transfer agreement; U.S. Patent Nos. 5,462,872 and 5,580,720). Sup-T1 cells were obtained from the NIH AIDS Research and Reference Reagent Program. BJAB cells and TF228.1.16 cells were maintained in TY medium [11] containing 10% fetal calf serum (FCS). Sup-T1 cells were maintained in RPMI 1640 supplemented with 10% FCS and penicillin–streptomycin (pen– strep). All cells were incubated at 377C and 5% CO2 . PBL were prepared by the usual Ficol-gradient separation from buffy coat samples (obtained from the NIH bloodbank) and cultured in RPMI 1640 at 377C and 5% CO2 for 24 h before further use. Subsequently, CD4/ cells were separated from PBL using a high-affinity negative selection column (R&D Systems, Minneapolis, MN). The resulting column eluate was washed once with serum-free RPMI 1640 and
INTRODUCTION
An early step in HIV-1 infection of T-lymphocytes is the recognition of the target cell receptor (CD4) by the gp120 subunit of the envelope glycoprotein (env) [1, 2]. Subsequent membrane fusion reactions, mediated by the gp41 subunit [3, 4] and coreceptors on the target cell [5, 6], introduce the genetic material of the virus into the cell. Infected cells with exposed env on their surface can fuse with additional CD4/ cells, eventually creating large, multinucleated syncytia [7]. Syncytium formation, in assays using virus-produc1 To whom correspondence and reprint requests should be addressed at Division of Applied Pharmacology Research, CDER, FDA, 8301 Muirkirk Road, Laurel, MD 20708. Fax: (301) 594-3037. E-mail: [email protected].
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then cells were maintained in RPMI 1640 supplemented with 10% Nu-serum (Collaborative Biomedical Products/Becton Dickinson Labware, Bedford, MA) and pen–strep. The CD4/ cell line PM1CD26H [12], used for the virus infectivity assay, was cultured in RPMI 1640 supplemented with 10% FCS, pen-strep, L-glutamine, and 10 mM Hepes. The monocytotropic HIV-1BaL [13] virus was from Advanced Biotechnologies (Columbia, MD). The T-cell line tropic HTLV-IIIMN virus [14] was from the AIDS Research and Reference Reagent Program, NIH. Dye retention and nonspecific transfer tests. For a fluorescent label to be useful for measuring fusion events it must be retained well in the original cell and show little spontaneous pickup by unlabeled cells. TF228.2.16 cells (Ç106/ml) were labeled with the indicated dye as recommended by the manufacturer and resuspended in PBS. Two tubes were prepared, one with labeled cells to evaluate dye retention and one with 50% labeled and 50% unlabeled cells to evaluate nonspecific transfer (e.g., dye leakage and reuptake). Cells were incubated at 377C and data of fluorescence intensities were collected by flow cytometry every 30 min. We evaluated the following dyes: rhodamine 123, NBD-phosphatidyl choline, PKH2 and PKH26 (Sigma), calcein-AM, CMFDA, BCECF, dihydroethidium, Fluo-3AM, Fura Red, DiQ, and DiO (Molecular Probes Inc., Eugene, OR). Cell-to-cell fusion assay. TF228.1.16 cells were chosen as the unlabeled target cells because they are partially adherent and spread out into an irregular shape. Env/ TF228.1.16 cells were agitated into suspension, adjusted to a concentration of 2 1 106 cells/ml in TY medium, and distributed at 100 ml/well into a 96-well plate (up to 24 centrally located wells per plate). The plates were then centrifuged at 1000 rpm for 1 min to produce a uniform monolayer and incubated for at least 1 h before adding fluorescently labeled CD4/ cells. SupT1 cells are nonadherent and maintain a spherical shape in the absence of a suitable fusion partner. CD4/ Sup-T1 and CD4/ PBL cells were fluorescently labeled as follows: 1 ml of calcein-AM (5 mM in DMSO) was added to 106 cells in 1 ml of serum-free RPMI, incubated for 30 min, washed 11, resuspended in serum-free RPMI 1640 (without calcein-AM), and incubated for an additional 30 min to allow complete conversion of the dye. CD4/ cells were adjusted to their final indicated concentrations in TY medium and 100 ml was added to each well containing a TF228.1.16 monolayer or to wells containing TY medium only (controls) for a final volume of 200 ml/well. For inhibition assays, compounds (see Reagents) were added to wells just before the addition of CD4/ cells. Then the plates were centrifuged at 1000 rpm for 1 min to bring the fusion partners into contact. Finally, the plates were incubated at 377C and 5% CO2 . Fluorescence image cytometry was performed on a central region of each well at the indicated times. Fluorescence image cytometry. A stage-scanning laser microscope (Model ACAS 570; Meridian Instruments, Okemos, MI) was used to measure changes in the distribution of the fluorescent dye, calcein, a fluorescein derivative with an absorption peak at 494 nm and an emission peak at 517 nm. The argon ion laser was tuned to the 488nm wavelength and the output power was adjusted to 200 mW. The scan strength was adjusted to 20% (of output power) using an acousto-optic modulator (AOM) and a 1% neutral density filter was placed in the optical path to produce a 0.4-mW beam prior to entering the optical path of an Olympus IMT-2 inverted microscope equipped with a standard ‘‘B’’ block filter set (490-nm bandpass, 500-nm dichroic mirror, and 515-nm longpass). The computer-controlled x–y scanning stage was moved to a central region of interest within each well to be analyzed and an area was scanned in a 180 1 180 step raster pattern of 5-mm increments, creating a 2-dimensional pseudocolored image with a log scale lookup table (LUT). These settings permitted the acquisition of a large enough area (0.9 1 0.9 mm) to visualize several syncytia, while maintaining a scan resolution sufficient to discriminate unfused Sup-T1 cells (diameter É15 mm) from cell debris. Labeled CD4/ cells only (control wells) were used to adjust the gain on the photomultiplier tube (PMT) to produce an
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image where the brightest pixels were within the maximum response range. Data analysis. The 2-dimensional pseudocolored images were processed to provide data on the following parameters: total area occupied by fluorescence, average area of contiguous fluorescent areas, and average shape factor of a contiguous fluorescent area. In all cases, a pixel value of 3 times background (determined from the pixel histogram of the nonfused control image) was used for the threshold value to subtract from the images. An average area per cell (nonfused controls) was calculated and one-half that value was used as the lower limit for subsequent size-range restrictions for the determination of average area and shape factor. Infection with cell-free virus suspension and reverse transcriptase (RT) assay. PM1-CD26H cells were incubated with serial dilutions of cytochalasin D (dissolved in DMSO), with 1% DMSO alone or with medium for 1 h at 377C. Cells were infected by mixing 2 1 105 cell/ ml with cell-free virus stocks yielding 1 1 105 cpm rA:dT RT activity/ ml. Four hours after initial infection, the cells were washed and cultured further with different dilutions of cytochalasin D or DMSO. The cultures were evaluated for syncytium formation, by microscopic examination, and for RT activity 4 days after infection. Supernatants of cultures were assayed in triplicate for RT activity using an exogenous template poly(rA:dT) as described previously by Oravecz et al. [12]. Levels of RT are expressed as the mean cpm of [3H]thymidine triphosphate incorporation into acid insoluble DNA.
RESULTS
Dye Retention and Secondary Uptake The results of the dye retention and secondary uptake tests for calcein-AM are shown in Fig. 1. The data clearly show that very little leakage of the dye in these cells occurs and that there is essentially no pickup of the leaked dye by the unlabeled cells. If a second color was needed, Fura Red performed nearly as well. Other tested dyes did not perform as well and had greater leakage rates, secondary uptake rates, or both. These
FIG. 1. Calcein retention within labeled TF228.1.16 cells. Mean fluorescence intensities at indicated time points calculated from flow cytometry histograms of calcein-labeled TF228.1.16 cells alone (open squares) or mixed 1:1 with unlabeled TF228.1.16 cells (labeled, open circles; unlabeled, closed circles).
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results should be regarded as valid for TF228.1.16 cells, other cell lines may behave differently. Time Course of Several Image Analysis Parameters during Cell-to-Cell Fusion Figure 2 illustrates the time-dependent changes in fluorescence localization using several ratios of fusion partners. At the 5-min time point the dye is concentrated within the area occupied by the labeled Sup-T1 cells and produces the greatest fluorescence intensity. As the labeled cells fuse with the underlying TF228.1.16 cells, the dye spreads into the cytoplasmic compartment of the target cells, increasing the area occupied by fluorescence while decreasing the intensity of the signal as the dye becomes more diffuse. Subsequent image analysis of these 2-dimensional pseudocolored images allowed us to monitor specific parameters that could be expected to change during the fusion process. By defining a threshold value, above which fluorescent pixels would be considered positive, the total area of the field occupied by cells containing the indicator dye could be determined. We chose a threshold value three times greater than background, as determined by the histogram of pixel intensities for a control image. As expected, the total area increases as labeled cells fuse with neighboring unlabeled cells (Fig. 3A). The lower Sup-T1 seeding densities (1 1 103 and 2 1 103) showed the greatest rate of change over the longest period. At higher densities, the syncytia initiated by individual labeled Sup-T1 cells begin to fuse with each other, resulting in very little net gain in fluorescence area. At later time points the largest syncytia lose their membrane integrity and the dye begins to leak out, resulting in a decrease in total area. Individual cells or syncytia can be identified by defining them as contiguous areas of fluorescence, surrounded by pixels below the threshold value. Those fluorescent areas at the edge of the scanned area may represent only part of a cell or syncytium. Therefore, fluorescent areas whose perimeter is partially defined by the border are eliminated from the image. Also, a size-range restriction can be applied to eliminate fluorescent debris. Here, we used a value of 75 mm2 as our lower limit, which is half the area of a normal labeled Sup-T1 cell. An upper limit of 106 mm2 was used since the syncytium could potentially occupy the entire scanned area (8.1 1 105 mm2). At this point, the average area of contiguous fluorescence or the average size of the syncytia can be calculated (Fig. 3B). The average area at 5 min corresponds to the average size of unfused Sup-T1 cells. The labeled cells fuse with the underlying TF228.1.16 cells and, subsequently, adjacent TF228.1.16 cells become included in the syncytium. Figure 3C shows the changes in the shape factor, or
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the ratio of the longest and shortest axes through the average fluorescent area, i.e., the roundness of the fluorescent object. Since the unfused Sup-T1 cells are spherical and appear circular in a 2-dimensional image scan, their shape factor equals 1 before they begin fusing. As the labeled cells fuse with the irregularly shaped TF228.1.16 cells, the absolute value of the ratio between the long and the short axes of the areas increases. Eventually, the larger syncytia begin to round up and return toward a shape factor of 1. Average area was chosen as the parameter on which to base the assay because total area appeared more sensitive to variations in the initial seeding densities of the labeled cells. Shape factor would be insufficient by itself in screening inhibitors, since large syncytia could become round. A low ratio of Sup-T1:TF228.1.16 cells (1:100) and an incubation period of 3 h were used in subsequent assays to reduce the contribution of syncytium-to-syncytium fusion events, allowing us to look at the progress of fusion from individual Sup-T1 cells or fusion origins. Fluorescently labeled Sup-T1 cells alone were used as unfused controls because they have approximately the same average area as cells seeded onto TF228.1.16 cells for õ5 min or those seeded onto BJAB cells, the untransfected parental cell line, at 3 h, thus allowing us to use only the two cell lines of the fusion pair and a single time point for scanning the multiwell plate. Effect of Various Anti-CD4 mAbs on the Cell-to-Cell Fusion Process Monoclonal antibodies were selected from several CD4-specific antibodies known to inhibit HIV binding and/or sycytium formation [15–18, reviewed in 19]. Anti-Leu3a, an antibody that binds to the CDR2 loop on the D1 domain of the CD4 molecule, effectively blocked fusion when TF228.1.16 cells were paired with Sup-T1 cells (Fig. 4A). When CD4/ human PBL cells were used as the fusion partner (Fig. 4B) the anti-Leu3a also blocked fusion at doses similar to those of Sup-T1 cells (Table 1). OKT4E, which binds opposite to the Leu3a epitope on the D1 domain, also blocked Sup-T1 fusion with TF228.1.16 cells in a dose-dependent manner (Fig. 4C). As the negative control, OKT4, an antibody that binds to the D4 domain, had no effect (not shown). Effect of Various Compounds on the Cell-to-Cell Fusion Process ATA was used as an example of a low-molecularweight inhibitor of nonimmunological origin. It has been shown to block anti-Leu3a binding to CD4 [20– 22] and to inhibit syncytium formation between HIV1-infected cells and uninfected CD4/ cells [23, 24]. ATA blocked fusion between TF228.1.16 and Sup-T1 cells in a dose-dependent manner, as shown in Fig. 5A. T20 (DP-178) was used as an example of a synthetic peptide
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FIG. 2. Pseudo-colored 2-dimensional image scans demonstrating time-dependent changes in fluorescence localization using different ratios of cell-to-cell fusion partners. Sup-T1 cells, labeled with calcein, were seeded at different densities onto a monolayer of TF228.1.16 cells. At the indicated time points, fluorescence data from a centralized region of interest from each well were collected using a stage-scanning laser microscope. Individual panels are representative of images collected from triplicate wells.
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inhibitor. It is derived from a putative helical domain within the transmembrane protein gp41 of HIV-1 and has been shown to be a potent anti-fusogenic molecule [25, 26]. T20 also blocked fusion in our assay (Fig. 5B). Cytochalasin D, in a concentration known to affect the microfilament system [27], also blocked fusion in a dose-dependent fashion (Fig. 5C). Table 1 summarizes
FIG. 3. Time-dependent changes in 2-dimensional image parameters. Cytometric analysis of fluorescence image scans was performed to derive the following parameters: (A) the total area of the field occupied by fluorescent pixels having a value greater than the threshold value; (B) the average area of contiguous fluorescent pixels surrounded by pixels below the threshold value; and (C) the shape factor, or the ratio of the longest and shortest axes through a contiguous fluorescent area. Triplicate image scans were taken at the indicated time points for the following Sup-T1 to TF228.1.16 cell ratios: 1:20 (solid line), 1:40 (dashed line), 1:100 (dashed-dotted line), and 1:200 (dotted line).
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FIG. 4. Inhibition of syncytia formation by mAbs directed against the CD4 molecule. CD4/ cells, labeled with calcein, were seeded onto a monolayer of TF228.1.16 cells and the average area of fluorescence was measured in the absence or presence of different concentrations of mAbs. Dose–response curves for (A) Sup-T1:TF228.1.16 fusion partners treated with anti-Leu3a, (B) CD4/ PBL:TF228.1.16 fusion partners treated with anti-Leu3a, and (C) Sup-T1:TF228.1.16 fusion partners treated with OKT4E. Each graph shows the results of one typical experiment, where each circle represents the value for 1 of the 24 wells. Open circles correspond to control fusion (upper dotted line) and control nonfusion (lower dashed line). Closed circles correspond to mAb treatment (solid line).
IC50 values for each of these compounds as well as the mAb values for comparison. Effect of Cytochalasin D on HIV Replication Cytochalasin D affected syncytia formation, but its activity to suppress HIV replication had not been de-
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TABLE 1 Fusion Inhibitors Ranked by Potency IC50a { SD (ng/ml)
Treatment Anti-Leu3ab Anti-Leu3a T20 Cytochalasin D OKT4E Aurintricarboxylic acid
6.1 7.1 25 130 800 12500
{ { { { { {
No. of experiments
2.1 2.7 11 47 100 1400
3 6 3 3 3 2
plane. The precision of the stage allows us to rescan the same region of interest ({1 mm) at various times after incubation. The acquisition time for each image is determined by the step size (or interpixel spacing) and the number of pixels in each direction, x and y. The particular combination we standardized on, 180 1 180 at 5 mm, requires 53 s to acquire an image, and
a Triplicate wells for each dose plus controls (see Fig. 4) were fitted to the Hill equation to produce IC50 values for each experiment. The average value of the nonfusion wells was used as the minimum response (0%) and the difference between the average value of the normal fusion wells and the nonfusion wells was used as the maximum response (100%). Experimental data were normalized to this range. Results are expressed as the mean IC50 ({SD) for multiple experiments. b CD4/ PBL was used instead of Sup-T1 cells as the fusion partner with TF228.1.16 cells.
scribed yet. Figure 6 shows that cytochalasin D significantly suppressed virus production, as measured by RT activity, of both monocytotropic (HIV-1BaL) and the Tcell line tropic (HTLV-IIIMN) viruses in PM1 cell cultures at the concentration of 0.1 mg/ml. The effect of cytochalasin D was dose dependent. The same dose dependency could be observed microscopically in those cultures at cytochalasin D doses of 0.1 and 1.0 mg/ml. At higher doses, especially at 10 mg/ml, cytochalasin D was toxic to the cells (not shown). DISCUSSION
The development of env expressing cell lines permits the evaluation of potential therapeutic compounds directed at the earliest steps of HIV infection, which are binding and fusion. Together with assays that can detect inhibition of CD4 binding using surrogates such as recombinant gp120 and anti-Leu3a mAb [21, 28, 29], the mechanism by which surface-acting compounds inhibit HIV infection can be better characterized. With this utility in mind we have developed a rapid, fluorescence-based, semiautomated cell-to-cell fusion assay. This fluorescence-based fusion assay was developed on an ACAS 570 stage-scanning laser microscope system. The laser beam is focused to irradiate a small spot (õ1 mm diameter) within the region of interest, an AOM modulates the beam to produce a short pulse of light (õ10 ms), and a sensitive PMT assigns a fluorescence intensity value to a specific x–y coordinate. These conditions permit fluorescence intensity assessment without significant bleaching of fluorescent indicators. The computer-controlled stage collects fluorescence data in a raster scanning pattern of the x–y
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FIG. 5. Inhibition of syncytia formation by various test compounds. Sup-T1 cells, labeled with calcein, were seeded onto a monolayer of TF228.1.16 cells and the average area of fluorescence was measured in the absence or presence of different concentrations of test compounds. Dose–response curves for (A) aurin tricarboxylic acid, (B) T20 peptide, and (C) cytochalasin D. Each graph shows the results of one typical experiment, where each circle represents the value for 1 of the 24 wells. Open circles correspond to control fusion (upper dotted line) and control nonfusion (lower dashed line). Closed circles correspond to test compounds (solid line).
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FIG. 6. Reverse transcriptase (RT) assay results of culture supernatants of PM1–CD26H cells infected with either the HIV-1Bal (open bars) or the HTLV-IIIMN (solid bars) virus. Results of the RT assays are expressed as the mean cpm of [3H]thymidine incorporation ({SD) into acid soluble DNA. (PM1–CD26H cells without virus, 106 { 5 cpm).
the time it takes to automatically move to the next region of interest and manually refocus brings the total time between images to Ç60 s. Although complete automation might be achieved with the introduction of autofocusing [30], and substantially reduce the operator time involved, it would not significantly reduce the total acquisition time for an experiment. Other fluorescence-based cell-to-cell fusion assays have been developed [31–33]. We chose to standardize our assay on the intracellular indicator dye, calcein, for the following reasons: (1) it is well retained, with essentially no nonspecific transfer (Fig. 1); (2) it still produces a detectable signal in the cells at the outer margins of the fusion process, where the intracellular concentration of dye is much more diffuse (Fig. 2); and (3) it is less likely to interfere with cell-surface events, as happens with the membrane soluble dye, PKH26 [32]. The anti-Leu3a mAb blocked cell-to-cell fusion in a dose-dependent fashion in both Sup-T1 cells and CD4/ cells separated from human PBL with approximately the same IC50 values (Figs. 4A and 4B and Table 1). OKT4E mAb also blocked fusion in a dose-dependent manner, although its IC50 was two orders of magnitude higher than that of anti-Leu3a (Fig. 4C and Table 1). OKT4E recognizes a discontinuous epitope within the D1 domain, and its relatively weak ability to interfere with the env-CD4 interaction has been attributed to indirect steric effects [18]. Other mAbs, which bind to epitopes even more distal to the gp120 binding site, have also been shown to block fusion without interfering with the binding of gp120 to the CD4 molecule. For example, mAb 5A8 binds to the D2 domain of CD4 and blocks fusion, presumably by restricting conforma-
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tional changes in CD4 and/or env [34, 35]. Healey et al. [36] also speculated that inhibition of conformational changes in CD4 was responsible for similar effects observed with Q425 and Q428, mAbs recognizing epitopes within the D3 domain of CD4. Interestingly, inhibition of fusion, using Sup-T1 cells pretreated with either OKT4E or Leu3a, could be completely reversed by anti-isotype antibodies (not shown). While the possibility exists that, for OKT4E (a mAb which does not block binding), the secondary antibody actually reduces the avidity of OKT4E sufficiently to permit env-induced conformational changes in CD4, it seems less likely that this would be the case for antiLeu3a (a mAb which blocks binding) [19]. Another possible explanation, consistent with the observations for both primary mAbs, is that capping of the crosslinked CD4 by the anti-isotype antibodies [37] leaves any unbound CD4 free to form env-CD4 multimers, which have been suggested as being necessary for fusion to occur [38]. To assess the usefulness of our assay, we tested several dissimilar compounds that could be expected to inhibit cell-to-cell fusion by different mechanisms. ATA presumably acts directly on CD4, the cell-surface receptor of the target cell, and is known to block the binding of HIV surrogates such as recombinant gp120 and antiLeu3a [21, 22] as well as alter the conformation of CD4 itself [39]. On the other hand, the T20 peptide inhibits fusion by interfering with the development of the proper configuration of gp41, the fusogenic component of the HIV envelope glycoprotein [4, 25, 26]. Both these compounds inhibited syncytia formation in a dose-dependent manner as determined by our assay (Figs. 5A, 5B and Table 1). Cytochalasin D does not affect anti-Leu3a mAb or gp120 binding at the doses used in our cell-to-cell fusion assay (not shown), but it does inhibit syncytia formation (Fig. 5C and Table 1). In fact, Frey et al. showed previously that cytochalasin D blocks fusion between gp160 expressing CHO and CD4 expressing Sup-T1 cells [40]. We have taken this finding further and have shown that cytochalasin D blocks T-cell-tropic and monocyotropic HIV replication, in a dose-dependent fashion, as determined by RT assay (Fig. 6). Cytochalasin D also inhibited syncytium formation, in a dosedependent fashion, in the very same cell cultures (not shown). We speculate, in agreement with Frey et al. [40], that an intact microfilament system plays a role in the cell-to-cell and virus-to-cell fusion by an unknown mechanism. Taken together, we have developed a semiautomated fluorescence-based cell-to-cell fusion assay for cells expressing either env or CD4. Quantitation is based on an increase of fluorescence area during the fusion event. The assay was shown to be useful for estimating IC50 values of inhibitory, CD4-directed mAbs and for
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compounds known to inhibit syncytia formation by three different mechanisms. Although the assay was developed on a laser-scanning microscope system, similar experiments could be performed using a conventional fluorescence microscope equipped with a lowlight-sensitive camera and computer software capable of morphometric analysis for area determinations. Furthermore, we suggest that the assay that we developed can be extended to measure fusion events in other pairs of fusion partners, provided that one of the cell types can be labeled with a bright, well-retained, cytoplasmic indicator dye. We thank Drs. Z. L. Jonak (SmithKline Beecham Pharmaceuticals) and P. E. Rao (Ortho Diagnostic Systems) for helpful discussions regarding cell lines and anti-CD4 monoclonal antibodies.
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lymphoid cells. In ‘‘Electromanipulation in Hybridoma Technology’’ (Borrebaeck and Hagen, Eds.), pp. 31–46, Stockton Press, New York. Oravecz, T., Roderiquez, G., Koffi, J., Wang, J., Ditto, M., BouHabib, D. C., Lusso, P., and Norcross, M. A. (1995). CD26 expression correlates with entry, replication and cytopathicity of monocytotropic HIV-1 strain in a T-cell line. Nat. Med. 1, 919– 926. Gartner, S., Markovits, P., Markovitz, D. M., Kaplan, M. H., Gallo, R. C., and Popovic, M. (1986). The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233, 215–219. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske, J., Safai, M., White, G., and Foster, P. (1984). Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 500– 503. Sattentau, Q. J., Dalgleish, A. G., Weiss, R. A., and Beverley, P. C. L. (1986). Epitopes of the CD4 antigen and HIV infection. Science 234, 1120–1123. Jameson, B. A., Rao, P. E., Kong, L. I., Hahn, B. H., Shaw, G. M., Hood, L. E., and Kent, S. B. (1988). Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein. Science 240, 1335–1339. Peterson, A., and Seed, B. (1988). Genetic analysis of monoclonal antibody and HIV binding sites on the human lymphocyte antigen CD4. Cell 54, 65–72. Mizukami, T., Fuerst, T. R., Berger, E. A., and Moss, B. (1988). Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 85, 9273–9277. Kieber-Emmons, T., Jameson, B. A., and Morrow, W. J. W. (1989). The gp120–CD4 interface: Structural, immunological and pathological considerations. Biochim. Biophys. Acta 989, 281–300. Schols, D., Baba, M., Pauwels, R., Desmyter, J., and De Clercq, E. (1989). Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor. Proc. Natl. Acad. Sci. USA 86, 3322–3326. Weaver, J. L., Gergely, P., Pine, P. S., Patzer, E., and Aszalos, A. (1990). Polyionic compounds selectively alter availability of CD4 receptors for HIV coat protein rgp120. AIDS Res. Hum. Retroviruses 6, 1125–1130. Chandler, G., Elcock, C., Depledge, P., Wrigley, S., Mous, J., Malkovsky, M., Moore, M., and Gammon, G. (1993). CD4-binding compounds: An assay to detect new classes of immunopharmacological agents. Int. J. Immunopharmacol. 15, 361–369. Cushman, M., Wang, P., Chang, S. H., Wild, C., De Clercq, E., Schols, D., Goldman, M. E., and Bowen, J. A. (1991). Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: Direct correlation of antiviral potency with molecular weight. J. Med. Chem. 34, 329–337. Cushman, M., Kanamathareddy, S., De Clercq, E., Schols, D., Goldman, M. E., and Bowen, J. A. (1991). Synthesis and antiHIV activities of low molecular weight aurintricaroxylic acid fragments and related compounds. J. Med. Chem. 34, 337–342. Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., and Matthews, T. J. (1994). Peptides corresponding to a predictive a-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc. Natl. Acad. Sci. USA 91, 9770–9774. Chen, C.-H., Matthews, T. J., McDanal, C. B., Bolognesi, D. P., and Greenberg, M. L. (1995). A molecular clasp in the human
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CELL-TO-CELL FUSION ASSAY immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: Implication for viral fusion. J. Virol. 69, 3771–3777. 27. Aszalos, A., Pine, P. S., Weaver, J. L., and Rao, P. E. (1994). Cytochalasin D modulates CD4 crosslinking sensitive mitogenic signal in T lymphocytes. Cell Immunol. 157, 81–91. 28. Weaver, J. L., Pine, P. S., Anand, R., Bell, S., and Aszalos, A. (1992). Inhibition of binding of HIV rgp120 to CD4 by dyes. Antiviral Chem. Chemother. 3, 147–151. 29. Weaver, J. L., Pine, P. S., Dutschman, G., Cheng, Y. C., Lee, K. H., and Aszalos, A. (1992). Prevention of binding of rgp120 by anti-HIV active tannins. Biochem. Biopharmacol. 43, 2479– 2480. 30. Price, J. H., and Gough, D. A. (1994). Comparison of phase-contrast and fluorescence digital autofocus for scanning microscopy. Cytometry 16, 283–297. 31. Dimitrov, D. S., Golding, H., and Blumenthal, R. (1991). Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes. Aids Res. Hum. Retroviruses 7, 799–805. 32. Dimitrov, D. S., and Blumenthal, R. (1994). Photoinactivation and kinetics of membrane fusion mediated by the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 68, 1956–1961. 33. Broder, C. C., Dimitrov, D. S., Blumenthal, R., and Berger, E. A. (1993). The block to HIV-1 envelope glycoprotein fusion in animal cells expressing human CD4 can be overcome by a human cell component(s). Virology 193, 483–491.
34. Burkly, L. C., Olson, D., Shapiro, R., Winkler, G., Rosa, J. J., Thomas, D. W., Williams, C., and Chisholm, P. (1992). Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. J. Immunol. 149, 1779–1787. 35. Moore, J. P., Sattentau, Q. J., Klasse, P. J., and Burkly, L. C. (1992). A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4/ cells. J. Virol. 66, 4784–4793. 36. Healey, D., Dianda, L., Moore, J. P., McDougal, J. S., Moore, M. J., Estess, P., Buck, D., Kwong, P. D., Beverly, P. C. L., and Sattentau, Q. J. (1990). Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4/ cells from vius binding. J. Exp. Med. 172, 1233–1242. 37. Kammer, G. M., Smith, J. A., and Mitchell, R. (1983). Capping of human T cell specific determinants: Kinetics of capping and receptor re-expression and regulation by the cytoskeleton. J. Immunol. 130, 38–44. 38. Earl, P. L., Doms, R. W., and Moss, B. (1992). Multimeric CD4 binding exhibited by human and simian immunodeficiency virus envelope protein dimers. J. Virol. 66, 5610–5614. 39. Szabo, G., Jr., Pine, P. S., Weaver, J. L., Rao, P. E., and Aszalos, A. (1992). CD4 changes conformation upon ligand binding. J. Immunol. 149, 3596–3609. 40. Frey, S., Marsh, M., Gunther, S., Pelchen-Matthews, A., Stephens, P., Ortlepp, S., and Stegmann, T. (1995). Temperature dependence of cell–cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1. J. Virol. 69, 1462–1472.
Received July 14, 1997 Revised version received December 22, 1997
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A Semiautomated Fluorescence-Based Cell-to-Cell Fusion Assay for gp120–gp41 and CD4 Expressing Cells P. Scott Pine,*,1 James L. Weaver,* Tamas Oravecz,† Marina Pall,† Michael Ussery,‡ and Adorjan Aszalos* *Division of Applied Pharmacology Research, CDER, Food and Drug Administration, Laurel, Maryland 20708; †Division of Hematologic Products, CBER, Food and Drug Administration, Bethesda, Maryland 20708; and ‡Antiviral Research Laboratory, CDER, Food and Drug Administration, Rockville, Maryland 20708
ing cells, has been used as an endpoint for measuring various inhibitors of HIV infection [1, 8]. Transfected cell lines, which express complementary components of the fusion reaction, can be used as models of cell-tocell fusion and avoid some of the problems associated with using live viruses [9, 10]. We present a quantitative assay based upon fluorescence image analysis of cell-to-cell fusion using CD4/ cells, either Sup-T1 cells or CD4/-enriched human peripheral blood lymphocytes (PBL), and env/ TF228.1.16 cells, BJAB cells which stably express the gp120–gp41 complex. This assay is performed in a multiwell plate and can be completed in less than a day. We show that changes in the two-dimensional distribution of the fluorescent indicator, calcein, can be used to measure cell-to-cell fusion and syncytium formation. The method was shown to yield quantitative results for prevention of syncytium formation by evaluating several compounds known to act at different sites in the HIV infection process.
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4/ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120–gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4/ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cellto-cell fusion. q 1998 Academic Press
MATERIALS AND METHODS Reagents. Aurin tricarboxylic acid (ATA) and cytochalasin D were from Sigma (St. Louis, MO). The peptide T20 was obtained from Dr. Dennis M. Lambert (Trimeris Inc., Research Triangle Park, NC) by material transfer agreement. T20 is a 36-amino-acid synthetic peptide derived from a domain within gp41. The monoclonal antibody (mAb) anti-Leu3a was from Becton Dickinson Immunocytometry (San Jose, CA); OKT4 and OKT4E were from Ortho Diagnostic Systems (Raritan, NJ). Cells and culture conditions. TF228.1.16 cells and BJAB cells were obtained from Dr. Z. Jonak at SmithKline Beecham Pharmaceuticals (by material transfer agreement; U.S. Patent Nos. 5,462,872 and 5,580,720). Sup-T1 cells were obtained from the NIH AIDS Research and Reference Reagent Program. BJAB cells and TF228.1.16 cells were maintained in TY medium [11] containing 10% fetal calf serum (FCS). Sup-T1 cells were maintained in RPMI 1640 supplemented with 10% FCS and penicillin–streptomycin (pen– strep). All cells were incubated at 377C and 5% CO2 . PBL were prepared by the usual Ficol-gradient separation from buffy coat samples (obtained from the NIH bloodbank) and cultured in RPMI 1640 at 377C and 5% CO2 for 24 h before further use. Subsequently, CD4/ cells were separated from PBL using a high-affinity negative selection column (R&D Systems, Minneapolis, MN). The resulting column eluate was washed once with serum-free RPMI 1640 and
INTRODUCTION
An early step in HIV-1 infection of T-lymphocytes is the recognition of the target cell receptor (CD4) by the gp120 subunit of the envelope glycoprotein (env) [1, 2]. Subsequent membrane fusion reactions, mediated by the gp41 subunit [3, 4] and coreceptors on the target cell [5, 6], introduce the genetic material of the virus into the cell. Infected cells with exposed env on their surface can fuse with additional CD4/ cells, eventually creating large, multinucleated syncytia [7]. Syncytium formation, in assays using virus-produc1 To whom correspondence and reprint requests should be addressed at Division of Applied Pharmacology Research, CDER, FDA, 8301 Muirkirk Road, Laurel, MD 20708. Fax: (301) 594-3037. E-mail: [email protected].
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then cells were maintained in RPMI 1640 supplemented with 10% Nu-serum (Collaborative Biomedical Products/Becton Dickinson Labware, Bedford, MA) and pen–strep. The CD4/ cell line PM1CD26H [12], used for the virus infectivity assay, was cultured in RPMI 1640 supplemented with 10% FCS, pen-strep, L-glutamine, and 10 mM Hepes. The monocytotropic HIV-1BaL [13] virus was from Advanced Biotechnologies (Columbia, MD). The T-cell line tropic HTLV-IIIMN virus [14] was from the AIDS Research and Reference Reagent Program, NIH. Dye retention and nonspecific transfer tests. For a fluorescent label to be useful for measuring fusion events it must be retained well in the original cell and show little spontaneous pickup by unlabeled cells. TF228.2.16 cells (Ç106/ml) were labeled with the indicated dye as recommended by the manufacturer and resuspended in PBS. Two tubes were prepared, one with labeled cells to evaluate dye retention and one with 50% labeled and 50% unlabeled cells to evaluate nonspecific transfer (e.g., dye leakage and reuptake). Cells were incubated at 377C and data of fluorescence intensities were collected by flow cytometry every 30 min. We evaluated the following dyes: rhodamine 123, NBD-phosphatidyl choline, PKH2 and PKH26 (Sigma), calcein-AM, CMFDA, BCECF, dihydroethidium, Fluo-3AM, Fura Red, DiQ, and DiO (Molecular Probes Inc., Eugene, OR). Cell-to-cell fusion assay. TF228.1.16 cells were chosen as the unlabeled target cells because they are partially adherent and spread out into an irregular shape. Env/ TF228.1.16 cells were agitated into suspension, adjusted to a concentration of 2 1 106 cells/ml in TY medium, and distributed at 100 ml/well into a 96-well plate (up to 24 centrally located wells per plate). The plates were then centrifuged at 1000 rpm for 1 min to produce a uniform monolayer and incubated for at least 1 h before adding fluorescently labeled CD4/ cells. SupT1 cells are nonadherent and maintain a spherical shape in the absence of a suitable fusion partner. CD4/ Sup-T1 and CD4/ PBL cells were fluorescently labeled as follows: 1 ml of calcein-AM (5 mM in DMSO) was added to 106 cells in 1 ml of serum-free RPMI, incubated for 30 min, washed 11, resuspended in serum-free RPMI 1640 (without calcein-AM), and incubated for an additional 30 min to allow complete conversion of the dye. CD4/ cells were adjusted to their final indicated concentrations in TY medium and 100 ml was added to each well containing a TF228.1.16 monolayer or to wells containing TY medium only (controls) for a final volume of 200 ml/well. For inhibition assays, compounds (see Reagents) were added to wells just before the addition of CD4/ cells. Then the plates were centrifuged at 1000 rpm for 1 min to bring the fusion partners into contact. Finally, the plates were incubated at 377C and 5% CO2 . Fluorescence image cytometry was performed on a central region of each well at the indicated times. Fluorescence image cytometry. A stage-scanning laser microscope (Model ACAS 570; Meridian Instruments, Okemos, MI) was used to measure changes in the distribution of the fluorescent dye, calcein, a fluorescein derivative with an absorption peak at 494 nm and an emission peak at 517 nm. The argon ion laser was tuned to the 488nm wavelength and the output power was adjusted to 200 mW. The scan strength was adjusted to 20% (of output power) using an acousto-optic modulator (AOM) and a 1% neutral density filter was placed in the optical path to produce a 0.4-mW beam prior to entering the optical path of an Olympus IMT-2 inverted microscope equipped with a standard ‘‘B’’ block filter set (490-nm bandpass, 500-nm dichroic mirror, and 515-nm longpass). The computer-controlled x–y scanning stage was moved to a central region of interest within each well to be analyzed and an area was scanned in a 180 1 180 step raster pattern of 5-mm increments, creating a 2-dimensional pseudocolored image with a log scale lookup table (LUT). These settings permitted the acquisition of a large enough area (0.9 1 0.9 mm) to visualize several syncytia, while maintaining a scan resolution sufficient to discriminate unfused Sup-T1 cells (diameter É15 mm) from cell debris. Labeled CD4/ cells only (control wells) were used to adjust the gain on the photomultiplier tube (PMT) to produce an
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image where the brightest pixels were within the maximum response range. Data analysis. The 2-dimensional pseudocolored images were processed to provide data on the following parameters: total area occupied by fluorescence, average area of contiguous fluorescent areas, and average shape factor of a contiguous fluorescent area. In all cases, a pixel value of 3 times background (determined from the pixel histogram of the nonfused control image) was used for the threshold value to subtract from the images. An average area per cell (nonfused controls) was calculated and one-half that value was used as the lower limit for subsequent size-range restrictions for the determination of average area and shape factor. Infection with cell-free virus suspension and reverse transcriptase (RT) assay. PM1-CD26H cells were incubated with serial dilutions of cytochalasin D (dissolved in DMSO), with 1% DMSO alone or with medium for 1 h at 377C. Cells were infected by mixing 2 1 105 cell/ ml with cell-free virus stocks yielding 1 1 105 cpm rA:dT RT activity/ ml. Four hours after initial infection, the cells were washed and cultured further with different dilutions of cytochalasin D or DMSO. The cultures were evaluated for syncytium formation, by microscopic examination, and for RT activity 4 days after infection. Supernatants of cultures were assayed in triplicate for RT activity using an exogenous template poly(rA:dT) as described previously by Oravecz et al. [12]. Levels of RT are expressed as the mean cpm of [3H]thymidine triphosphate incorporation into acid insoluble DNA.
RESULTS
Dye Retention and Secondary Uptake The results of the dye retention and secondary uptake tests for calcein-AM are shown in Fig. 1. The data clearly show that very little leakage of the dye in these cells occurs and that there is essentially no pickup of the leaked dye by the unlabeled cells. If a second color was needed, Fura Red performed nearly as well. Other tested dyes did not perform as well and had greater leakage rates, secondary uptake rates, or both. These
FIG. 1. Calcein retention within labeled TF228.1.16 cells. Mean fluorescence intensities at indicated time points calculated from flow cytometry histograms of calcein-labeled TF228.1.16 cells alone (open squares) or mixed 1:1 with unlabeled TF228.1.16 cells (labeled, open circles; unlabeled, closed circles).
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results should be regarded as valid for TF228.1.16 cells, other cell lines may behave differently. Time Course of Several Image Analysis Parameters during Cell-to-Cell Fusion Figure 2 illustrates the time-dependent changes in fluorescence localization using several ratios of fusion partners. At the 5-min time point the dye is concentrated within the area occupied by the labeled Sup-T1 cells and produces the greatest fluorescence intensity. As the labeled cells fuse with the underlying TF228.1.16 cells, the dye spreads into the cytoplasmic compartment of the target cells, increasing the area occupied by fluorescence while decreasing the intensity of the signal as the dye becomes more diffuse. Subsequent image analysis of these 2-dimensional pseudocolored images allowed us to monitor specific parameters that could be expected to change during the fusion process. By defining a threshold value, above which fluorescent pixels would be considered positive, the total area of the field occupied by cells containing the indicator dye could be determined. We chose a threshold value three times greater than background, as determined by the histogram of pixel intensities for a control image. As expected, the total area increases as labeled cells fuse with neighboring unlabeled cells (Fig. 3A). The lower Sup-T1 seeding densities (1 1 103 and 2 1 103) showed the greatest rate of change over the longest period. At higher densities, the syncytia initiated by individual labeled Sup-T1 cells begin to fuse with each other, resulting in very little net gain in fluorescence area. At later time points the largest syncytia lose their membrane integrity and the dye begins to leak out, resulting in a decrease in total area. Individual cells or syncytia can be identified by defining them as contiguous areas of fluorescence, surrounded by pixels below the threshold value. Those fluorescent areas at the edge of the scanned area may represent only part of a cell or syncytium. Therefore, fluorescent areas whose perimeter is partially defined by the border are eliminated from the image. Also, a size-range restriction can be applied to eliminate fluorescent debris. Here, we used a value of 75 mm2 as our lower limit, which is half the area of a normal labeled Sup-T1 cell. An upper limit of 106 mm2 was used since the syncytium could potentially occupy the entire scanned area (8.1 1 105 mm2). At this point, the average area of contiguous fluorescence or the average size of the syncytia can be calculated (Fig. 3B). The average area at 5 min corresponds to the average size of unfused Sup-T1 cells. The labeled cells fuse with the underlying TF228.1.16 cells and, subsequently, adjacent TF228.1.16 cells become included in the syncytium. Figure 3C shows the changes in the shape factor, or
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the ratio of the longest and shortest axes through the average fluorescent area, i.e., the roundness of the fluorescent object. Since the unfused Sup-T1 cells are spherical and appear circular in a 2-dimensional image scan, their shape factor equals 1 before they begin fusing. As the labeled cells fuse with the irregularly shaped TF228.1.16 cells, the absolute value of the ratio between the long and the short axes of the areas increases. Eventually, the larger syncytia begin to round up and return toward a shape factor of 1. Average area was chosen as the parameter on which to base the assay because total area appeared more sensitive to variations in the initial seeding densities of the labeled cells. Shape factor would be insufficient by itself in screening inhibitors, since large syncytia could become round. A low ratio of Sup-T1:TF228.1.16 cells (1:100) and an incubation period of 3 h were used in subsequent assays to reduce the contribution of syncytium-to-syncytium fusion events, allowing us to look at the progress of fusion from individual Sup-T1 cells or fusion origins. Fluorescently labeled Sup-T1 cells alone were used as unfused controls because they have approximately the same average area as cells seeded onto TF228.1.16 cells for õ5 min or those seeded onto BJAB cells, the untransfected parental cell line, at 3 h, thus allowing us to use only the two cell lines of the fusion pair and a single time point for scanning the multiwell plate. Effect of Various Anti-CD4 mAbs on the Cell-to-Cell Fusion Process Monoclonal antibodies were selected from several CD4-specific antibodies known to inhibit HIV binding and/or sycytium formation [15–18, reviewed in 19]. Anti-Leu3a, an antibody that binds to the CDR2 loop on the D1 domain of the CD4 molecule, effectively blocked fusion when TF228.1.16 cells were paired with Sup-T1 cells (Fig. 4A). When CD4/ human PBL cells were used as the fusion partner (Fig. 4B) the anti-Leu3a also blocked fusion at doses similar to those of Sup-T1 cells (Table 1). OKT4E, which binds opposite to the Leu3a epitope on the D1 domain, also blocked Sup-T1 fusion with TF228.1.16 cells in a dose-dependent manner (Fig. 4C). As the negative control, OKT4, an antibody that binds to the D4 domain, had no effect (not shown). Effect of Various Compounds on the Cell-to-Cell Fusion Process ATA was used as an example of a low-molecularweight inhibitor of nonimmunological origin. It has been shown to block anti-Leu3a binding to CD4 [20– 22] and to inhibit syncytium formation between HIV1-infected cells and uninfected CD4/ cells [23, 24]. ATA blocked fusion between TF228.1.16 and Sup-T1 cells in a dose-dependent manner, as shown in Fig. 5A. T20 (DP-178) was used as an example of a synthetic peptide
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FIG. 2. Pseudo-colored 2-dimensional image scans demonstrating time-dependent changes in fluorescence localization using different ratios of cell-to-cell fusion partners. Sup-T1 cells, labeled with calcein, were seeded at different densities onto a monolayer of TF228.1.16 cells. At the indicated time points, fluorescence data from a centralized region of interest from each well were collected using a stage-scanning laser microscope. Individual panels are representative of images collected from triplicate wells.
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inhibitor. It is derived from a putative helical domain within the transmembrane protein gp41 of HIV-1 and has been shown to be a potent anti-fusogenic molecule [25, 26]. T20 also blocked fusion in our assay (Fig. 5B). Cytochalasin D, in a concentration known to affect the microfilament system [27], also blocked fusion in a dose-dependent fashion (Fig. 5C). Table 1 summarizes
FIG. 3. Time-dependent changes in 2-dimensional image parameters. Cytometric analysis of fluorescence image scans was performed to derive the following parameters: (A) the total area of the field occupied by fluorescent pixels having a value greater than the threshold value; (B) the average area of contiguous fluorescent pixels surrounded by pixels below the threshold value; and (C) the shape factor, or the ratio of the longest and shortest axes through a contiguous fluorescent area. Triplicate image scans were taken at the indicated time points for the following Sup-T1 to TF228.1.16 cell ratios: 1:20 (solid line), 1:40 (dashed line), 1:100 (dashed-dotted line), and 1:200 (dotted line).
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FIG. 4. Inhibition of syncytia formation by mAbs directed against the CD4 molecule. CD4/ cells, labeled with calcein, were seeded onto a monolayer of TF228.1.16 cells and the average area of fluorescence was measured in the absence or presence of different concentrations of mAbs. Dose–response curves for (A) Sup-T1:TF228.1.16 fusion partners treated with anti-Leu3a, (B) CD4/ PBL:TF228.1.16 fusion partners treated with anti-Leu3a, and (C) Sup-T1:TF228.1.16 fusion partners treated with OKT4E. Each graph shows the results of one typical experiment, where each circle represents the value for 1 of the 24 wells. Open circles correspond to control fusion (upper dotted line) and control nonfusion (lower dashed line). Closed circles correspond to mAb treatment (solid line).
IC50 values for each of these compounds as well as the mAb values for comparison. Effect of Cytochalasin D on HIV Replication Cytochalasin D affected syncytia formation, but its activity to suppress HIV replication had not been de-
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TABLE 1 Fusion Inhibitors Ranked by Potency IC50a { SD (ng/ml)
Treatment Anti-Leu3ab Anti-Leu3a T20 Cytochalasin D OKT4E Aurintricarboxylic acid
6.1 7.1 25 130 800 12500
{ { { { { {
No. of experiments
2.1 2.7 11 47 100 1400
3 6 3 3 3 2
plane. The precision of the stage allows us to rescan the same region of interest ({1 mm) at various times after incubation. The acquisition time for each image is determined by the step size (or interpixel spacing) and the number of pixels in each direction, x and y. The particular combination we standardized on, 180 1 180 at 5 mm, requires 53 s to acquire an image, and
a Triplicate wells for each dose plus controls (see Fig. 4) were fitted to the Hill equation to produce IC50 values for each experiment. The average value of the nonfusion wells was used as the minimum response (0%) and the difference between the average value of the normal fusion wells and the nonfusion wells was used as the maximum response (100%). Experimental data were normalized to this range. Results are expressed as the mean IC50 ({SD) for multiple experiments. b CD4/ PBL was used instead of Sup-T1 cells as the fusion partner with TF228.1.16 cells.
scribed yet. Figure 6 shows that cytochalasin D significantly suppressed virus production, as measured by RT activity, of both monocytotropic (HIV-1BaL) and the Tcell line tropic (HTLV-IIIMN) viruses in PM1 cell cultures at the concentration of 0.1 mg/ml. The effect of cytochalasin D was dose dependent. The same dose dependency could be observed microscopically in those cultures at cytochalasin D doses of 0.1 and 1.0 mg/ml. At higher doses, especially at 10 mg/ml, cytochalasin D was toxic to the cells (not shown). DISCUSSION
The development of env expressing cell lines permits the evaluation of potential therapeutic compounds directed at the earliest steps of HIV infection, which are binding and fusion. Together with assays that can detect inhibition of CD4 binding using surrogates such as recombinant gp120 and anti-Leu3a mAb [21, 28, 29], the mechanism by which surface-acting compounds inhibit HIV infection can be better characterized. With this utility in mind we have developed a rapid, fluorescence-based, semiautomated cell-to-cell fusion assay. This fluorescence-based fusion assay was developed on an ACAS 570 stage-scanning laser microscope system. The laser beam is focused to irradiate a small spot (õ1 mm diameter) within the region of interest, an AOM modulates the beam to produce a short pulse of light (õ10 ms), and a sensitive PMT assigns a fluorescence intensity value to a specific x–y coordinate. These conditions permit fluorescence intensity assessment without significant bleaching of fluorescent indicators. The computer-controlled stage collects fluorescence data in a raster scanning pattern of the x–y
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FIG. 5. Inhibition of syncytia formation by various test compounds. Sup-T1 cells, labeled with calcein, were seeded onto a monolayer of TF228.1.16 cells and the average area of fluorescence was measured in the absence or presence of different concentrations of test compounds. Dose–response curves for (A) aurin tricarboxylic acid, (B) T20 peptide, and (C) cytochalasin D. Each graph shows the results of one typical experiment, where each circle represents the value for 1 of the 24 wells. Open circles correspond to control fusion (upper dotted line) and control nonfusion (lower dashed line). Closed circles correspond to test compounds (solid line).
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FIG. 6. Reverse transcriptase (RT) assay results of culture supernatants of PM1–CD26H cells infected with either the HIV-1Bal (open bars) or the HTLV-IIIMN (solid bars) virus. Results of the RT assays are expressed as the mean cpm of [3H]thymidine incorporation ({SD) into acid soluble DNA. (PM1–CD26H cells without virus, 106 { 5 cpm).
the time it takes to automatically move to the next region of interest and manually refocus brings the total time between images to Ç60 s. Although complete automation might be achieved with the introduction of autofocusing [30], and substantially reduce the operator time involved, it would not significantly reduce the total acquisition time for an experiment. Other fluorescence-based cell-to-cell fusion assays have been developed [31–33]. We chose to standardize our assay on the intracellular indicator dye, calcein, for the following reasons: (1) it is well retained, with essentially no nonspecific transfer (Fig. 1); (2) it still produces a detectable signal in the cells at the outer margins of the fusion process, where the intracellular concentration of dye is much more diffuse (Fig. 2); and (3) it is less likely to interfere with cell-surface events, as happens with the membrane soluble dye, PKH26 [32]. The anti-Leu3a mAb blocked cell-to-cell fusion in a dose-dependent fashion in both Sup-T1 cells and CD4/ cells separated from human PBL with approximately the same IC50 values (Figs. 4A and 4B and Table 1). OKT4E mAb also blocked fusion in a dose-dependent manner, although its IC50 was two orders of magnitude higher than that of anti-Leu3a (Fig. 4C and Table 1). OKT4E recognizes a discontinuous epitope within the D1 domain, and its relatively weak ability to interfere with the env-CD4 interaction has been attributed to indirect steric effects [18]. Other mAbs, which bind to epitopes even more distal to the gp120 binding site, have also been shown to block fusion without interfering with the binding of gp120 to the CD4 molecule. For example, mAb 5A8 binds to the D2 domain of CD4 and blocks fusion, presumably by restricting conforma-
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tional changes in CD4 and/or env [34, 35]. Healey et al. [36] also speculated that inhibition of conformational changes in CD4 was responsible for similar effects observed with Q425 and Q428, mAbs recognizing epitopes within the D3 domain of CD4. Interestingly, inhibition of fusion, using Sup-T1 cells pretreated with either OKT4E or Leu3a, could be completely reversed by anti-isotype antibodies (not shown). While the possibility exists that, for OKT4E (a mAb which does not block binding), the secondary antibody actually reduces the avidity of OKT4E sufficiently to permit env-induced conformational changes in CD4, it seems less likely that this would be the case for antiLeu3a (a mAb which blocks binding) [19]. Another possible explanation, consistent with the observations for both primary mAbs, is that capping of the crosslinked CD4 by the anti-isotype antibodies [37] leaves any unbound CD4 free to form env-CD4 multimers, which have been suggested as being necessary for fusion to occur [38]. To assess the usefulness of our assay, we tested several dissimilar compounds that could be expected to inhibit cell-to-cell fusion by different mechanisms. ATA presumably acts directly on CD4, the cell-surface receptor of the target cell, and is known to block the binding of HIV surrogates such as recombinant gp120 and antiLeu3a [21, 22] as well as alter the conformation of CD4 itself [39]. On the other hand, the T20 peptide inhibits fusion by interfering with the development of the proper configuration of gp41, the fusogenic component of the HIV envelope glycoprotein [4, 25, 26]. Both these compounds inhibited syncytia formation in a dose-dependent manner as determined by our assay (Figs. 5A, 5B and Table 1). Cytochalasin D does not affect anti-Leu3a mAb or gp120 binding at the doses used in our cell-to-cell fusion assay (not shown), but it does inhibit syncytia formation (Fig. 5C and Table 1). In fact, Frey et al. showed previously that cytochalasin D blocks fusion between gp160 expressing CHO and CD4 expressing Sup-T1 cells [40]. We have taken this finding further and have shown that cytochalasin D blocks T-cell-tropic and monocyotropic HIV replication, in a dose-dependent fashion, as determined by RT assay (Fig. 6). Cytochalasin D also inhibited syncytium formation, in a dosedependent fashion, in the very same cell cultures (not shown). We speculate, in agreement with Frey et al. [40], that an intact microfilament system plays a role in the cell-to-cell and virus-to-cell fusion by an unknown mechanism. Taken together, we have developed a semiautomated fluorescence-based cell-to-cell fusion assay for cells expressing either env or CD4. Quantitation is based on an increase of fluorescence area during the fusion event. The assay was shown to be useful for estimating IC50 values of inhibitory, CD4-directed mAbs and for
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compounds known to inhibit syncytia formation by three different mechanisms. Although the assay was developed on a laser-scanning microscope system, similar experiments could be performed using a conventional fluorescence microscope equipped with a lowlight-sensitive camera and computer software capable of morphometric analysis for area determinations. Furthermore, we suggest that the assay that we developed can be extended to measure fusion events in other pairs of fusion partners, provided that one of the cell types can be labeled with a bright, well-retained, cytoplasmic indicator dye. We thank Drs. Z. L. Jonak (SmithKline Beecham Pharmaceuticals) and P. E. Rao (Ortho Diagnostic Systems) for helpful discussions regarding cell lines and anti-CD4 monoclonal antibodies.
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Received July 14, 1997 Revised version received December 22, 1997
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