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Arthritis Center, Department of Medicine, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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500 kDa and a fraction between 50 and 500 kDa.
Following ultracentrifugation, the activity in the 500-kDa fraction
was concentrated in the pellet (FasL vector preparation (VP)), which
was also infective when added to NIH-3T3 cells. Both Polybrene and
poly-L-lysine significantly enhanced the cytotoxicity of
FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and
cell-associated FasL. In the presence of Polybrene, FasL VP killed
targets that are resistant to anti-Fas mAb and sFasL. The
infectivity but not FasL cytotoxicity of FasL VP was sensitive to
irradiation and heat shock. By contrast, cytotoxicity of FasL VP could
be enhanced or inhibited depending on the doses of anti-FasL mAb.
Interestingly, the infectivity of FasL VP was specifically enhanced by
anti-FasL mAb, suggesting that a nonviral gene product could be
used to regulate the behavior of the retroviral vector. Thus, in
addition to expressing potent FasL cytotoxicity, the FasL VP exhibits
unique properties heretofore not attributed to anti-Fas mAb, sFasL,
and cell-associated FasL. Our study raises the possibility of using the
retroviral gene-packaging technology to make powerful, versatile, and
regulatable bioactive vesicles expressing a predetermined function of
the protein encoded by the target gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to cell-associated FasL, sFasL, and anti-Fas mAb, bioactive vesicles expressing FasL has also been described (13, 14, 15). In most cases, the vesicles expressed extremely weak FasL-mediated cytotoxicity (13, 14). We have observed a potent FasL-mediated cytotoxicity in the retroviral vector prepared from a packaging cell line that carries the human FasL gene (29). The FasL vector preparation (FasL VP) is made of a family of particles including active vectors, inactive vectors, and vesicles. Because the packaging cell line over-expresses both the viral proteins and the FasL proteins, FasL VP is expected to express these proteins as well. Our initial study using Western blotting and immuno-adsorption has demonstrated the presence of FasL protein and Fas-specific cytotoxicity in the FasL VP, including the expression of FasL protein by the active vectors (29). The data suggest that the retroviral gene transfer technology can be used to produce bioactive vesicles displaying predetermined apoptotic function encoded by the target gene, i.e., fasl. In the present study, we demonstrate that FasL VP exhibited several distinctive properties heretofore not attributed to cell-associated FasL, sFasL, and anti-Fas mAb. We show that the gene transfer function of the FasL VP could be dissociated from the cytotoxicity mediated directly by FasL VP. Although these bioactivities of FasL VP could be dissociated, the infectivity of retroviral vector was significantly enhanced by the presence of anti-FasL mAb. Thus, by introducing a cell-associated protein into retroviral vector, it may be possible to regulate the vector function and behavior through the protein encoded by the nonviral gene. Our study has significant implication in gene transfer technology and gene therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The retroviral vector for human FasL gene was produced according to the method of Miller et al. (16). The human FasL cDNA expression construct was kindly provided by Dr. S. Nagata (Osaka University Medical Center, Osaka, Japan; Ref. 7). The human FasL (hFasL) cDNA was cloned into pLXSN (American Type Culture Collection (ATCC), Manassas, VA), transfected into PE501 cells by Lipofectamine (Life Technologies-MRL, Gaithersburg, MD). PE501 cells were obtained from Dr. A. D. Miller (Fred Hutchinson Cancer Institute, Seattle, WA). After culturing the transiently transfected cells for 24 h, culture supernatants laden with ecotropic virus were obtained and used to infect the PA317 Moloney murine leukemia virus packaging cell line (PA317 MoMLV, also from A. D. Miller) to generate the amphotropic vectors. Clones were selected with culture medium containing G418 (0.4 mg/ml). Clones were expanded using the same selection medium. Six clones were tested and all expressed strong FasL cytotoxicity. One line that expressed stronger cytotoxicity than the others was selected for further study. Experiments periodically conducted with other clones are consistent with those described herein. A similarly prepared packaging cell line carrying the human cKrox gene (Krox-PA317), which codes for the transcription factor Krox, was used as control throughout the study (17).
Preparation of vector-containing and vector-free fractions
The hFasL-PA317 and the Krox-PA317 cells were first cultured in the presence of G418 to 70% confluence. The medium was replaced with culture medium without G418 and cultured for 2 days. The supernatant was collected for further study, and the cells were used as effectors in cytotoxicity assays. The supernatant was centrifuged at 13,000 rpm for 30 min to remove cell debris and then centrifuged at 25,000 rpm for 17 h with a Beckman ultracentrifuge (Beckman, Fullerton, CA). The top 80% volume of the supernatant was collected as vector-free supernatant (VFS). The pellet, used as FasL VP, was obtained in parallel by a 3-h centrifugation under otherwise identical condition. The vector-containing pellet was suspended in medium and then sterilized by passing through a 0.45-micron filter. The sFasL was also obtained by fractionation of cell-free supernatant (CFS) using a Millipore Centricon-500 (Millipore, Bedford, MA) that filters through proteins with a m.w. <500 kDa. The molecular size of the sFasL fraction was estimated with Millipore Centricon-100 and Millipore Centricon-50, which separate proteins with a m.w. <100 kDa and 50 kDa, respectively.
FasL cytotoxicity
Seven tumor cell lines were used as targets for FasL
cytotoxicity. The human T lymphoma cell line Jurkat expresses human Fas
and is sensitive to FasL. LB27.4 is a mouse B lymphoma hybridoma
(ATCC), which expresses mouse Fas and is sensitive to both mouse and
human FasL (Ref. 18 , and this study).
LF+ and LF- targets are a
pair of mouse T lymphomas that have been transfected with mouse Fas
gene and antisense of Fas gene such that the former is sensitive and
the latter is completely resistant to FasL (9). The
lpr-derived T cell hybridoma 5D5, which lacks Fas, was also
used for specificity control (19). A macrophage hybridoma,
m
59 (20), and a T cell hybridoma, K31H28
(21), both highly resistant to Jo2 anti-Fas mAb
(PharMingen, San Diego, CA), were used to demonstrate the potency of
FasL VP as a cytotoxic agent. Target cells (2 x
104), labeled with
Na251CrO4
as previously described (22), were mixed with various
cytotoxic reagents including activated 5D5 T cell hybridoma cells,
which express cytotoxic FasL following incubation with plate-bound
anti-CD3 mAb (19). After culture for 5 h, culture
supernatants were collected and the radioactivity released to the
supernatant was counted with a gamma-counter. Cells cultured in the
absence of cytotoxic reagent were used as background controls. The
radioactivity released by cells cultured in the presence of 0.5%
Nonidet P-40 was used as a reference for total cell death. The
cytotoxic activity, expressed as % specific
51Cr-release, was determined by the formula:
[(cpm of sample - cpm of background)/(cpm of total release
- cpm of background)] x 100%. In some experiments, the effect of
enhancers or inhibitors on cytotoxicity was tested by the addition of
inhibitors at the beginning of culture. The enhancers tested were
Polybrene and poly-L-lysine (Sigma, St. Louis, MO). The
inhibitors tested were Fas-Ig (21) and NOK-1 anti-FasL
mAb (PharMingen).
Effect of irradiation and heat shock on virus infectivity and FasL cytotoxicity
FasL VP was either treated with 10,000 rads by gamma-irradiation with a 137Cs irradiator or heat-shocked at various temperatures for 30 min in a water bath. Treated FasL VP was then tested for the ability to transfer G418 resistance to NIH-3T3 cells and the ability to kill Fas+ targets. Both the FasL VP not subject to irradiation and the FasL VP incubated in a 37°C water bath were examined in parallel. The infection activity was determined by counting the number of growing G418-resistant colonies according to the method of Miller et al. (16). The results were expressed as total CFU per sample preparation. Cytotoxicity of each sample was determined as described above and expressed as lytic units per sample preparation. A lytic unit is defined as the amount of cytotoxic materials, volume, or cell number required to cause 20% specific lysis of LB27.4 target in the 5-h 51Cr-release assay.
Effect of NOK-1 anti-FasL mAb on FasL VP cytotoxicity and infectivity
The dose of FasL VP that induced 35–55% killing of LB27.4 in a
5-h assay was used to determine the effect of NOK-1 anti-FasL mAb
(IgG1 isotype, PharMingen) on FasL VP cytotoxicity. The experiments
were conducted by adding various doses of NOK-1 at the beginning of the
assays. An irrelevant hybridoma protein of the IgG1 isotype was
included as a control. The effect of NOK-1 on viral infectivity was
determined as follows: We titrated the FasL VP to determine the amount
capable of generating 
70 colonies in a well (6-well plate) plated
with 2 x 104 NIH-3T3 cells 1 day earlier.
Various concentrations of NOK-1 or control isotype were cultured with
FasL VP (50 µl) during the 6 h of infection. Wells were washed
three times with medium and then cultured in medium containing 0.75
mg/ml of G418. The number of growing G418-resistant colonies was
determined 10 days later by Giemsa stain (Sigma). The results were
expressed as number of colonies per well.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The retroviral packaging cell line, hFasL-PA317, which carries the
human FasL gene, was tested for FasL-mediated cytotoxicity against a
panel of targets including both human (Jurkat) and mouse (LB27.4 and
LF+) tumor cells. The parental PA317 and the
control Krox-PA317 packaging cells were used for comparison. In
addition, the mouse T cell hybridoma 5D5 (19), which lacks
Fas and expresses FasL-mediated cytotoxicity upon stimulation with
immobilized anti-CD3, was included as a positive control. The
results indicate that hFasL-PA317 cells express potent cytotoxicity
against Jurkat, LB27.4, and LF+ targets that
express Fas (Fig. 1
A).
LF- targets, which lack surface Fas, were
resistant (Fig. 1A). In contrast, cytotoxicity was not
observed with the Krox-PA317 cells. Soluble Fas-Ig but not a
nonspecific human IgG1 myeloma protein inhibited the cytotoxicity
against LB27.4 targets (Fig. 1B). The cytotoxicity expressed
by hFasL-PA317 cells was comparable to or stronger than 5D5 T cells
that were activated by plate-bound anti-CD3 (Fig. 1A).
Other sensitive targets included human T leukemia cells MOLT-4 and
U266B1 myeloma (ATCC) (data not shown). These observations demonstrate
that hFasL-PA317 cells express FasL-mediated cytotoxicity against a
large panel of Fas+ targets of both human and
murine origins.
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We reasoned that there could be three components in the
hFasL-PA317 cells capable of expressing FasL-mediated cytotoxicity. One
is the FasL expressed on the cells, the second is sFasL released by
metalloproteinase cleavage, and the third is FasL associated with the
FasL VP. To demonstrate the various forms of FasL-mediated
cytotoxicity, a graded number of the hFasL-PA317 cells were cultured in
a 96-well plate overnight. The adherent cells were washed with medium,
and target cells were added. The mixtures were cultured for 5 h,
and then the cytotoxicity was determined. Four replica plates were also
made but without the addition of labeled target cells. Supernatants
collected from one replica plate after a 5-h culture were mixed with
the labeled LB27.4 target and cultured for an additional 5 h.
There was little or no cytotoxicity detected with the supernatant
preparations. In contrast, strong killing of the same target cells was
observed with the hFasL-PA317 cells in a manner that was dependent on
E:T ratios (Fig. 2A). The data
demonstrate that under the defined experimental conditions, target
cells were killed by FasL associated with hFasL-PA317 and that, within
a 5-h culture period, any cytotoxic component that may have been
released was insufficient to account for the cytotoxicity of
hFasL-PA317 cells.
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B).
Therefore, the FasL VP prepared from 48-h culture supernatants were
used throughout the rest of this study.
For large quantity preparations, cells were cultured in large petri
dishes (150 mm x 25 mm; Falcon, Lincoln Park, NY). At 70%
confluence, culture medium was replaced with medium without G418.
Culture supernatant was collected 48 h later, and cell debris was
removed by centrifugation at 13,000 rpm for 30 min using a Sorvall
(Wilmington, DE) SS-34 rotor. The CFS collected was further centrifuged
at 25,000 rpm for 17 h at 5°C using SW-30 rotor in a Beckmann
ultracentrifuge. The top 80% of the supernatant was carefully removed
and used as VFS. This preparation lacked the viral vector as determined
by the inability of the supernatant to render the NIH-3T3 cells
resistant to G418 through viral infection (Fig. 3A). The pellet (FasL VP) was
suspended to 7% of the original volume, filtered through a 0.45-micron
membrane, and tested for infectivity.
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A). The low (40%) recovery of the ability to
infect NIH-3T3 cells was presumably due to retroviral inactivation
during ultracentrifugation or loss during filtration. These
preparations were also tested for FasL-mediated cytotoxicity (Fig. 3B). All fractions were able to kill LB27.4 targets in a 5-h
assay. The total cytotoxic activity present in the VFS was about the
same as the FasL VP. The combined cytotoxic activity of VFS and FasL VP
accounts for 65–80% of the activity of the original CFS. It should be
noted that the total lytic units present in the CFS is 15% of that
associated with hFasL-PA317 cells (Fig. 3 legend).
To further characterize the FasL activity, the CFS, the VFS, and the
FasL VP were fractionated with Millipore Centricon-500, which retains
components larger than 500 kDa. The results indicate that most of the
cytotoxic activity in the VFS was present in the components <500 kDa,
i.e., sFasL (Fig. 4A). Further
fractionation with Centricon-100 and Centricon-50 indicates nearly 30%
of the activity was confined between 50 and 100 kDa, and the remaining
activity was between 100 and 500 kDa. By contrast, the majority of the
cytotoxicity of the FasL VP was present in the compartment that
retained components larger than 500 kDa (Fig. 4B). A
comparable level of cytotoxic activity was observed in both
compartments when CFS was fractionated with Centricon-500 (Fig. 4C). However, the ability to transfer G418 resistance was
detected only in the fraction containing components larger than 500 kDa
(data not shown).
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Because FasL VP possesses the ability to transfect cells as
well as to kill the targets, it is conceivable that an efficient FasL
gene transfer could have caused or contributed to the expression of
FasL cytotoxicity. We used three methods to rule out this possibility
(Fig. 5). First, targets were treated
with actinomycin D for 45 min and then examined for sensitivity to FasL
VP. This treatment essentially inhibited to completion the gene
transcription in target cells (18) but had no inhibitory
effect on their sensitivity to FasL VP (Fig. 5A). Next, we
irradiated FasL VP with 10,000 rads and then examined FasL cytotoxicity
and the ability to transfer G418 resistance to NIH-3T3 cells. The
results indicate that the gamma-irradiation inhibited 64% the ability
of the viral vector to transfer G418 resistance, but had no effect on
its ability to kill Fas+ target cells (Fig. 5, B and C). The third method we tested was
heat-shock treatment. FasL VP were treated for 30 min at temperature
between 47°C and 75°C. FasL VP treated at 37°C was used as
control. The results showed that treatments at temperature below 75°C
had little effect on FasL VP cytotoxicity (Fig. 5D). A
moderate inhibition was observed with FasL VP treated at 75°C. In
sharp contrast, the heat-shock treatment completely eliminated the
ability of the virus to infect NIH-3T3 cells and transfer of G418
resistance (Fig. 5E). The data clearly demonstrate that
infection and FasL-mediated cytotoxicity could be dissociated and that
FasL gene transfer is not required for the FasL VP to express
FasL-mediated cytotoxicity.
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A well-established property of retroviral vector is the enhanced
efficiency of gene transfer by the presence of Polybrene, presumably by
increasing the contact between the viral vectors and host cells.
Therefore, the effect of Polybrene on the FasL-mediated cytotoxicity by
FasL VP was examined. The results showed that the cytotoxicity of FasL
VP was significantly increased by Polybrene in a dose-dependent manner
(Fig. 6A). Under the
experimental conditions, the cytotoxicity of FasL VP was increased as
the concentrations of Polybrene raised from 0 to 1.2 µg/ml. The
optimal enhancement of cytotoxicity was observed with Polybrene
concentrations ranged between 1.2 and 6 µg/ml. The enhancing activity
of Polybrene was not observed with the cytotoxicity of VFS,
hFasL-PA317, or Jo2 anti-Fas mAb (Fig. 6A). The ability
of Polybrene to increase the cytotoxicity of FasL VP appears to be due
to its multiple cationic characteristics because the same results were
obtained with poly-L-lysine (Fig. 6B). Similar
to Polybrene, poly-L-lysine enhanced the cytotoxicity of
FasL VP but not VFS, hFasL-PA317, or Jo2 anti-Fas mAb. The
enhancement of cytotoxicity varies among different targets. At the
extreme situation, targets (K31H28 T cell hybridoma and m59
hybridoma) that are resistant to high doses of Jo2 anti-Fas mAb and
the FasL VP became sensitive to FasL VP when Polybrene was added to the
culture (Fig. 7). Both K3 and m59 were
moderately sensitive to hFasL-PA317, Polybrene did not enhance the
killing (data not shown). Moreover, even in the presence of Polybrene,
FasL VP-mediated cytotoxicity could not be against two Fas-negative
targets, LF- and 5D5 (Fig. 7). These
observations indicate that the Polybrene-enhanced cytotoxicity was
mediated through Fas receptor, but the cytotoxicity of FasL VP can be
regulated in a fashion distinct from that of sFasL, Jo2 anti-Fas
mAb, and the membrane FasL associated with cells.
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The anti-FasL mAb NOK-1 has been shown to inhibit
FasL-mediated cytotoxicity (11); therefore, its effect on
the FasL VP-mediated cytotoxicity and transfer of G418 resistance was
determined. The FasL-PA317 cells, which express cell-associated FasL,
and VFS, which contains sFasL, were included as controls. Polybrene was
not used in this experiment. The results are shown in Fig. 8. When high doses of NOK-1 were used,
the cytotoxicity of all three samples was strongly inhibited. However,
when low doses of NOK-1 were used, the cytotoxicity of FasL VP, but not
hFasL-PA317 or VFS, was significantly enhanced.
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The effect of NOK-1 on gene transfer was studied in parallel. The
assays were conducted in the absence of Polybrene. The ability to
infect NIH-3T3 was not affected by the presence of 0.01–1 µg/ml of
NOK-1 (Fig. 9). Interestingly, the
ability to transfer G418-resistance was significantly enhanced when
infection was conducted in the presence of 2–10 µg/ml of NOK-1. No
effect on infectivity was observed when experiments were conducted with
an isotype control mAb. Under the experimental condition, the FasL VP
did not induce death of NIH-3T3 cells in a 5-h cytotoxicity assay,
suggesting that the enhancement was unlikely due to protection of cell
death by NOK-1. The data are consistent with the interpretation that
FasL vectors were cross-linked by NOK-1 and became more effective
presumably because of increased interactions between the cross-linked
vectors and cells. The results suggest strongly that the FasL of the
vector preparation can be used as a target to regulate viral
infectivity and gene transfer function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the retrovirus was generated from a packaging cell line, which carries the human FasL gene, it is conceivable that Fas-specific cytotoxicity is the result of gene transfer-induced expression of FasL in target cells, which in turn kills the Fas-expressing cells. Three pieces of evidence support the conclusion that the FasL VP directly expresses FasL-mediated cytotoxicity without the need for FasL gene transfer. First, inhibition of target cell transcription with actinomycin D did not inhibit the cytotoxicity of FasL VP. Second, both irradiation and heat-shock treatment inhibited the ability of the FasL VP to infect the host cells, which are more resistant to FasL (discussed below), but not its ability to kill sensitive targets. Third, full-length FasL protein was demonstrated in the FasL VP by Western blotting (29). However, under different conditions, FasL VP could transfer the FasL gene and to endow the recipient cells with FasL cytotoxicity (discussed below).
A unique property of FasL VP is its distinct response to polycationic molecules. Polybrene has been widely used to promote the efficiency of retroviral infection. Both Polybrene and poly-L-lysine enhance the cytotoxicity of FasL VP. The mechanism of the enhancement appears to be dependent on the interactions between FasL VP and target cells, which is increased by these polycationic molecules. The inability of sFasL to express higher cytotoxicity in the presence of Polybrene or poly-L-lysine is consistent with this interpretation. The inability of Polybrene to increase the cytotoxic activity of hFasL-PA317 suggests that the size of FasL-expressing membrane particles maybe another influencing factor. It is possible that hFasL-PA317 cells are sufficiently large such that they are unable to be cross-linked by Polybrene. Another explanation is that cellular FasL are more efficient in cross-linking once cell (E:T) interaction begins because of the higher number of FasL and the fluidity of the cell membrane in comparison to the small retroviral particles.
An interesting observation is the ability of low concentrations of
NOK-1 anti-FasL mAb to enhance the cytotoxicity of FasL VP but not
sFasL or FasL on hFasL-PA317, whereas high doses inhibit the
cytotoxicity of all three. Several anti-FasL mAbs have been shown
to inhibit cell-mediated cytotoxicity (11). We have found
four different mAbs to be able to enhance cytotoxicity of FasL VP
(S. Jodo, unpublished observation). The extremely low
concentration of the mAb (0.1–10 ng/ml) that was needed for
enhancement is remarkable. It suggests that this enhancement is unique
to FasL VP. The enhancement is consistent with the interpretation that
a proper cross-linking of vectors by NOK-1 increases the interactions
between vectors and target cells. However, additional factors must be
involved because NOK-1 mAb recognizes both sFasL and FasL on
hFasL-PA317 cells because their activity could be inhibited by a high
dose of NOK-1 (Fig. 8). Yet enhancement of cytotoxicity by lower doses
of NOK-1 was not observed.
The association of FasL cytotoxicity with the FasL VP also poses a
unique situation with respect to gene transfer, which is the original
purpose for generating such a vector. Because it kills Fas-expressing
cells, the target range for FasL gene transfer is limited to cells that
either are more resistant to the FasL cytotoxicity or can generate
FasL-resistance rapidly through selection. Consistent with the former
possibility, we have been unable to generate G418-resistant cells by
standard vector-mediated gene transfer experiments using LB27.4,
Jurkat, and LF+ targets. By contrast, when
NIH-3T3 and m59 cells were treated with FasL VP, G418-resistant
cells were generated. In contrast to their parental cells (NIH-3T3 and
m59), the G418-resistant cells express FasL cytotoxicity (indicating
FasL VP contains functional vectors for FasL gene), greatly reduce cell
surface Fas expression, and become highly resistant to FasL VP. The
G418-resistant, FasL-expressing NIH-3T3 cells produce FasL-expressing
vesicles but not viral particles (data not shown), indicating that the
production of bioactive vesicles expressing FasL cytotoxicity is not
dependent on the production of viral particles. The FasL cytotoxicity
of the vesicles produced by the G418-resistant NIH-3T3 cells was about
10% of that produced by the hFasL-PA317 packaging cells, raising
the possibility that active production of virus particles
facilitates the production of functional bioactive vesicles. In this
respect, it should be noted that the FasL cytotoxicity of FasL VP
prepared from hFasL-PA317 cells is 1000 times stronger than that
produced by PHA-activated Jurkat cells (data not shown), which has been
reported to produce bioactive vesicles expressing FasL function
(14).
The generation of bioactive vesicles from the hFasL-PA317 packaging cell line implies that other specific functions could also be produced. We have successfully generated similar retroviral vector cell line (TRAIL-PA317) carrying the human TRAIL protein. TRAIL VP prepared from TRAIL-PA317 kills Jurkat target in a TRAIL-specific fashion (29). Retroviral packaging cell lines, which carry other types of cell membrane proteins, are now being made to determine whether their vector preparations are capable of carrying out the specific functions of the proteins encoded by the target genes. For example, one could produce vector preparations from packaging cell lines carrying TNF or CD40 ligand and determine whether they can modulate fibroblast function or stimulate B cells, respectively (23, 24). In contrast to this protocol, modifications of the env gene have been used to express new functions on the retroviral particles, including redirecting target range and increase of virus/cell interaction (25, 26). It should be possible to use this approach to generate FasL VP with high target specificity. It is relevant that the vector’s ability to infect host cells could be eliminated completely by heat shock treatment, allowing the vector preparation to specifically and directly express the desired effector function of the protein in the absence of gene transfer.
The ability of NOK-1 to enhance the infectivity of FasL VP is of great significance because it indicates that a cell-derived protein (FasL) can have an effect on gene transfer when present in the vector preparation. The ability of anti-HIV Abs to enhance HIV infection in human cells has been demonstrated and the enhancement was mediated through the Fc receptor (27). How NOK-1 anti-FasL mAb enhances infectivity of the FasL VP remains to be elucidated. Because fibroblasts express little FcR (28), it seems unlikely that the enhancement by NOK-1 mAb is mediated through FcR. One possibility is that anti-FasL protects NIH-3T3 cells from FasL VP cytotoxicity, and hence increases the overall frequency of infected cells. If this interpretation is correct, then the actual infection efficiency of the FasL VP will be underestimated. However, FasL VP was not apparently cytotoxic to NIH-3T3 cells when cultured together for 5 h. We favor the interpretation that cross-linking of FasL VP by NOK-1 enhances the interaction between vectors and cells. This interpretation is consistent with the results obtained with Polybrene treatment, which enhances the infectivity and modulates the FasL cytotoxicity of FasL VP. The significance of our finding is that a protein encoded by the nonviral gene could be used to regulate the behavior of retroviral vectors, thus having important implications in studies using gene transfer and gene therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Arthritis Center, K508, Boston University School of Medicine, 71 East Concord Street, Boston, MA 02118.
3 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; VP, vector preparation; hFasL, human FasL; VFS, vector-free supernatant; CFS, cell-free supernatant.
Received for publication December 3, 1999. Accepted for publication March 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Gene Ther. 4:1181.[Medline]
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S. Jodo, A. M. Hohlbaum, S. Xiao, D. Chan, D. Strehlow, D. H. Sherr, A. Marshak-Rothstein, and S.-T. Ju CD95 (Fas) Ligand-Expressing Vesicles Display Antibody-Mediated, FcR-Dependent Enhancement of Cytotoxicity J. Immunol., November 15, 2000; 165(10): 5487 - 5494. [Abstract] [Full Text] [PDF]
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).
B, Supernatants of three additional replica plates were
collected at various times after culture and tested in a 5-h
cytotoxicity against 51Cr-labeled LB27.4. The cytotoxicity
of supernatant was slightly underestimated because 150 µl instead of
200 µl were collected for assays.
