(Investigative Ophthalmology and Visual Science. 2000;41:3134-3141.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Local Immunosuppression Prolongs Survival of RPE Xenografts Labeled by Retroviral Gene Transfer
Chi–Chun Lai1,5,
Peter Gouras1,
Kentaro Doi1,
Stephen H. Tsang2,3,
Stephen P. Goff2 and
Paul Ashton4
From the
1 Departments of Ophthalmology and
2 Biochemistry and Molecular Biophysics, Columbia University, Howard Hughes Medical Institute, New York, New York;
3 The Jules Stein Eye Institute, University of California Los Angeles; and
4 Tufts New England Medical Center, Boston, Massachusetts;
5 Chang Gung Memorial Hospital, Taipei, Taiwan.
 |
Abstract
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|---|
PURPOSE. To determine whether local immunosuppression with Cyclosporin A can
influence the survival of human fetal retinal pigment epithelium (RPE)
xenografts in the rabbit’s subretinal space.
METHODS. Cultured human fetal RPE cells were transduced with the gene for green
fluorescent protein (GFP) using a lentiviral vector. The RPE was
transplanted into the subretinal space of rabbits that received
intravitreal cyclosporine either by weekly injections (0.25–0.5 mg) or
by slow release (approximately 2 µg/d) from a capsule sutured into
the vitreal cavity after prior cryopexy. The transplanted RPE was
followed by GFP fluorescence scanning laser ophthalmoscopy and by
histology of the transplant site.
RESULTS. RPE xenografts in eyes receiving intravitreal cyclosporine survived
longer (several months) than they did in control eyes without
cyclosporine. Survival was as long with slow release capsules as it was
with weekly intravitreal injections at much higher concentrations of
cyclosporine.
CONCLUSIONS. Local immunosuppression of the eye with cyclosporine prolongs the
survival of RPE xenografts in the subretinal space of rabbits, implying
that rejection involves activated T lymphocytes. Local
immunosuppression with slow release capsules is as effective as weekly
injections at much higher concentrations.
|
Introduction
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|---|
One problem with retinal pigment epithelium (RPE) transplantation
is that the graft seems to be rejected in the subretinal space, even
though this is considered an immunologically privileged site. Systemic
immunosuppression can counteract such rejection, but this has
complicating side effects, especially in older subjects in whom
transplantation might have its most therapeutic applicability. One way
to circumvent systemic complications is to immunologically suppress
only the eye in which the transplant has been placed. Intravitreally
placed slow release capsules have been shown to deliver therapeutic
levels of the immunosuppressive drug cyclosporine over relatively long
periods of time.1
This may provide a way to prevent
rejection in the subretinal space without generalized
immunosuppression.
We have tested local immunosuppression of the eye, using RPE xenografts
in the subretinal space of rabbits by tracking the transplants with a
reporter gene encoding green fluorescent protein (GFP) introduced into
these cells by retroviral gene transfer. Such labeled transplants can
be followed noninvasively with the scanning laser ophthalmoscope (SLO),
which greatly facilitates carrying out such experiments because it
provides a means of continuously reexamining the transplants in vivo.
An abstract2
and a brief report3
of this
research have been published.
|
Methods
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|---|
Human fetal RPE was cultured and transduced with the gene for GFP
using a lentiviral vector.3
The virus was generated by
cotransfection of human kidney–derived 293T cells by three
plasmids.3
4
The packaging construct contained the
cytomegalovirus (CMV) promoter and the insulin polyadenylation signal
to express all the viral proteins in trans, except the envelope and
Vpu. The second plasmid provided a vector with all the
cis-acting elements that allow transfer and integration into
the target cells. In this transducing vector, an expression cassette
with the Rev responsive element and the CMV promoter is used to direct
the expression of GFP. The third plasmid provides the envelope protein
from the vesicular stomatitis virus glycoprotein to enhance the virus
stability and the range of possible targets. The lentiviral titers were
determined by infection of 293T cells seeded in six-well plates at
1 x 105 cells/well the day before infection
with serial dilution of the viral stock in the presence of 8 µg/ml
polybrene. After overnight incubation, the culture medium was changed
and the cells incubated for 2 more days. Fluorescent microscopy and/or
a fluorescence-activated cell sorter (FACStar plus; Becton–Dickinson,
Mountain View, CA) identified GFP fluorescent cells. Titers ranged from
107 to 109. The cultures
were exposed to the virus only once.
GFP fluorescent fetal human RPE was transplanted as a suspension,
approximately 50,000 cells in 50 microliters, into the subretinal space
of rabbits, anesthetized with ketamine (20 mg/kg intramuscularly
[IM]) and rompun (10 mg/kg, IM). The cells selected for
transplantation were followed in culture after transduction with
lentivirus to assess their GFP fluorescence. The percentage of cells
expressing GFP ranged from 40% to 80%. The transplanted cells were
not sorted for fluorescence before transplantation but were used
directly after they were removed from the culture plate by
trypsinization, washed with balanced salt solution, and concentrated by
gentle centrifugation. A glass pipette with a tip diameter of 100 to
150 µm was used to inject the cell solution subretinally under
microscopic control. Twenty-two rabbits received a transplant,
occupying a space less than a millimeter in diameter, in each eye
adjacent to the myelinated region of the optic nerve, which provides a
landmark to find the transplant site by SLO.
Fourteen rabbits received an intravitreal injection of Cyclosporine A
(CSA) weekly. Five rabbits received 0.25 mg/wk and nine
rabbits 0.5 mg/wk. The solutions were obtained by volumetrically
diluting a stock solution of CSA, 50 mg/ml. This is a standard solution
for the intravenous administration of cyclosporine, which contains 0.65
g/ml castor oil as a solvent and 33% ethyl alcohol as a preservative.
The opposite eye received an intravitreal injection of an equal volume
of a balanced salt solution in the controls.
Eight rabbits were used to study the effect of slow release capsules
containing only cyclosporine. The slow release capsule was sutured
through the sclera at the pars plana 2 weeks after local cryopexy. Slow
release capsules have been found to deliver approximately 2 µg of
cyclosporine daily into the vitreal chamber.1
The other
eye received a device consisting of polymers only without cyclosporine.
The rabbits were followed weekly by biomicroscopy, indirect
ophthalmoscopy, and scanning laser ophthalmoscopy (SLO) using infrared,
red and argon blue light, and fluorescence filtering for fluorescein
emission. Survival of the transplant was judged by the presence of GFP
fluorescence in the transplant area. When no fluorescence was detected,
the transplant was considered to have been rejected.3
At this point the rabbit was killed and the eyes removed for histology.
Three rabbits were killed at 2 to 3 weeks after all fluorescence had
disappeared. One rabbit was euthanatized while there was still strong
GFP fluorescence, and this rabbit was not considered in the survival
data. The eyes were fixed in either 3% buffered glutaraldehyde or 4%
paraformaldehyde in phosphate-buffered saline at pH 7.2. The former
eyes were processed for Epon embedding and thin sectioning and the
latter for cryosectioning and fluorescence microscopy. For
cryomicroscopy, the paraformaldehyde-fixed sections were immersed in
ornithine carbamoyltransferase compound and frozen by dry ice.
Sectioning was performed on a Leica 1850 cryotome (Leica Instruments,
Nusslach, Germany). Sections were mounted on gelatinized glass slides
with fluoromount-G and examined by fluorescence microscopy.
The animals were treated in conformity with the Declaration of
Helsinki, the Guiding Principles in the Care and Use of Animals (DHEW
Publication, NIH 80-23), and the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research.
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Results
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Figure 1A
shows a patch of human fetal RPE in culture viewed by white light
transillumination. The patch (asterisk) is heavily pigmented with
nondividing RPE. Along the edge of the patch migrating, dividing cells
can be seen with much less pigmentation (arrowheads). Figure 1B
shows
the patch of RPE in the presence of an additional strong blue light
that induces GFP fluorescence. Despite their dense pigmentation, the
fluorescence is intense enough to make GFP-expressing cells quite
visible. Some cells (small arrows) are fluorescing more strongly than
others. Many cells are fluorescing too dimly to be seen in the presence
of the transilluminating white light but can be identified and counted
by fluorescence illumination alone. The fraction of GFP-fluorescing
cells in this patch was estimated to be approximately 70%. The
fraction of GFP fluorescence obtained by lentiviral transduction varied
from 40% to 80%. This fluorescence remained stable in
vitro.3
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Figure 1. Color photographs showing a patch of human fetal RPE in vitro
(A). The darkly pigmented cells within the patch
(asterisk) have not divided; lightly pigmented, migrating
cells are visible along the edge of the patch (arrowheads).
(B) The same patch viewed with both fluorescence and
transillumination. Green fluorescence of GFP is evident in a
large proportion of the stationary cells in the patch. Some cells
(arrows) fluoresce more strongly than others. (C)
A frozen section of rabbit retina shows GFP fluorescent
xenografts on the host RPE layer. There is a weak yellowish
fluorescence considered to be lipofuscin surrounding black
cytoplasmic melanin in the host RPE (arrow). (D)
A frozen section of rabbit retina, viewed with both fluorescence
and transillumination, shows GFP-fluorescent xenografts on top of
the nonfluorescent host RPE layer (arrow at the basal margin of
this layer). (E) An Epon section of a transplant site,
which shows rejection. There is a dense concentration of monocytes
within the subretinal space (asterisk) and the adjacent
choroid (C) directly under a disrupted host RPE layer (an arrow
shows the basal edge of this layer). The neural retina
(NR) has few monocytes. The inflammatory
response is extremely local to this area of retina.
(F) An Epon section of a transplant site, which shows
another example of rejection. There are two foci of monocytes in the
subretinal space (asterisks) and the adjacent choroid (C).
An arrow shows the basal edge of the host RPE layer. The
neural retina (NR) has virtually no monocytes.
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Figure 2
shows the appearance of a transplant site in an eye with a slow release
capsule of cyclosporine at 2, 3, 4, and 12 weeks after surgery. The
upper set of photographs shows the appearance of the transplant in blue
light. The transplant site is a darker structure just to the left of
the highly reflecting myelinated nerve fibers at the optic nerve head
of the rabbit. There are several small, highly reflecting structures
close to the perimeter of the transplant that are not always visible
because they depend in large part on the angle of illumination (e.g.,
they are not very visible in the photograph at 4 weeks after
transplantation surgery). The lower set of photographs in Figure 2
shows the fluorescent appearance of the retina. Here relatively small,
bright structures about the size of single epithelial cells (i.e.,
approximately 10–30 µm) can be seen within and along the perimeter
of the transplant. They do not correspond to the reflecting structures
seen along the perimeter of the transplant in blue light, described
previously. These GFP fluorescent structures remained similar in size,
position, and brightness for the entire 12-week period after
transplantation.
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Figure 2. SLO views of a transplant site at 2, 3, 4, and 12 weeks after
transplantation seen by reflected blue light (upper set) and
fluorescence (lower set). The transplant, located adjacent
to the myelinated optic nerve fibers, is seen as darker area surrounded
by a light demarcation line in blue light and in fluorescence as a
structure containing bright pointlike structures, which are considered
to be GFP-expressing transplanted RPE. This eye had a slow release
capsule of cyclosporine.
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|
Figure 3
shows a transplant in the opposite eye, in which there was only a
polymer device without cyclosporine. The transplant site is a darker
elliptical structure, which has a dark vitreal band extending to the
retinotomy. The transplant is adjacent to the myelinated optic nerve,
seen on the right in the photograph (arrow) taken at 12 weeks after
transplantation surgery. The lower set of photographs in Figure 3
shows
the fluorescent appearance of the retina. At 2 weeks after
transplantation, numerous small fluorescent cell-like spots can be seen
throughout the transplant site. At 3 weeks after transplantation, many
of these fluorescent spots have disappeared. At 4 weeks after
transplantation, all the fluorescent spots have disappeared, which was
our criterion for rejection.3
This is also the appearance
at 12 weeks, although at that point some fluorescence is seen adjacent
the transplant site. These fluorescent spots are slightly larger, less
bright, and more amorphous than the GFP fluorescence. These structures
show a yellow fluorescence in histologic sections and appear as
lipofuscin-like particles in the host RPE layer by electron microscopy.
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Figure 3. SLO views of the transplant site in the opposite eye of that shown in
Figure 2
. This eye had a capsule without cyclosporine, and the
transplant shows evidence of rejection at 3 weeks. The upper
row shows the transplant in reflected blue light; the
transplant is a darker structure surrounded by a lighter demarcation
line. It is adjacent to the myelinated optic nerve fiber layer seen
only in the right-most photograph (arrowhead). The
lower row shows the fluorescent view. At 2 weeks,
GFP-fluorescing cells are visible within the transplant. At 3 weeks,
the number of fluorescing cells is reduced. At 4 weeks all fluorescence
has disappeared. At 12 weeks there is also no fluorescence within the
transplant; some lipofuscin-like fluorescence has developed outside the
transplant site (at the upper left).
|
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Figure 4
shows a transplant that has survived for 14 weeks after
transplantation. In the upper set of photographs, the reflected blue
light image shows the transplant site adjacent to the myelinated optic
nerve. The dark spot on the right edge of the transplant is the
retinotomy. In the lower set of photographs, the fluorescence of the
retina shows many brightly fluorescent spots, about the size of single
retinal epithelial cells, within the transplant site. These fluorescent
spots remain similar in size, shape, position, and brightness over
time. This eye received 0.25 mg/wk of cyclosporine intravitreally.
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Figure 4. SLO views of a transplant site in an eye receiving weekly injections of
cyclosporine (0.25 mg). The upper row shows the retina
in reflected blue light. The transplant is visible by a faint
demarcation line and mottling of the RPE layer; the retinotomy site is
seen as a dark spot adjacent to the myelinated optic nerves on the
right side of each photograph. The lower row
shows the fluorescent view. GFP fluorescent cells can be seen for 14
weeks after transplantation.
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Figure 5
illustrates a transplant that also survived for more than 7 weeks after
transplantation in an eye that contained a slow release capsule of
cyclosporine. In this case the retina is shown in blue, fluorescence,
and infrared images. The blue light image (upper set) shows the
transplant site below the myelinated optic nerve fibers. There is a
vitreal band extending to the retinotomy. The transplant site is
visible as a horizontal structure with a light demarcation line around
it. The fluorescent view (middle set) shows the GFP fluorescent
transplant cells within the transplant site. These cells retain their
shape, position, and brightness for the entire 7-week period. The
infrared view (lower set) reveals a dark band extending through the
entire transplant site. This darkness is considered to represent the
added density of the transplanted RPE cells within the subretinal
space, only a fraction of which exhibit GFP fluorescence.
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Figure 5. SLO views of a transplant site in an eye with a slow release capsule
containing cyclosporine at 2, 3, and 7 weeks after transplantation. The
upper row shows the retina in reflected blue light, the
middle row in fluorescence, and the lower
row in infrared light. GFP-fluorescing cells can be seen for
the entire time. The infrared view shows a darker reflected image
within the transplant site produced by the increase due to the
xenograft.
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|
This hypothesis was supported by the histology where transplanted cells
identified by their GFP fluorescence could be seen on top of the host
RPE layer (Figs. 1C
1D)
. When rejection occurred, GFP fluorescent
cells were no longer found in the retina, and when the transplant site
was examined within several weeks after the fluorescence had
disappeared, there was always clear evidence of rejection within the
transplant site. The rejection was characterized by intense collections
of monocytes in the choroid and focal areas of host RPE and
photoreceptor damage (Figs. 1E
1F)
. Many more monocytes were always
found in the choroid adjacent to the transplant site than in the neural
retina. The longer the time between the disappearance of the GFP
fluorescence in the retina and the euthanatization of the rabbit, the
less was the amount of monocytes detectable around the transplant site.
In some cases, the major evidence for rejection was disruption of the
host RPE layer and loss of photoreceptors at the transplant site.
Figure 6
compares the survival of the xenografts in the presence and absence of
local cyclosporine immunosuppression. Based on the criterion of
complete loss of GFP fluorescence as an indicator of rejection, locally
immunosuppressed transplants survived longer than those without
immunosuppression. Fifty percent of the RPE xenografts survived for at
least 5 weeks when associated with local immunosuppression. Without
immunosuppression, 50% of the xenografts disappeared in less than 1
month. The mean graft survival time for the controls, slow release CSA,
and 0.25 and 0.5 mg CSA weekly groups was 4, 11, 10, and 9 weeks,
respectively. The slow release of cyclosporine was as effective as
repeated intravitreal injections of much greater concentrations.
Survival of a transplant in one eye did not appear to be related to its
survival in the other eye. One RPE xenograft survived for at least 10
weeks without immunosuppression, whereas all the others disappeared
within 5 weeks. The loss of GFP fluorescence occurred relatively
quickly, disappearing within the course of 1 week. In some cases, there
was a more gradual loss of fluorescence, and this seemed more common in
the transplants that survived the longest. All the transplants
surviving for 15 weeks or longer had lost much of the fluorescence they
originally had.
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Figure 6. The survival time of each transplant as judged by the complete absence
of GFP fluorescence at the transplant site. The ordinate represents the
percentage of transplants that survived, and the abscissa represents
the time after transplantation surgery in weeks. The controls received
no CSA immunosuppression. The other three groups received either 0.25
or 0.5 mg CSA by weekly intravitreal injection or by a slow release
vitreal capsule.
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|
|
Discussion
|
|---|
These results indicate that local administration of cyclosporine
prolongs the survival of human fetal RPE xenografts in the subretinal
space of rabbits. This supports the hypothesis that classic rejection
plays a role in the survival of foreign transplants in the subretinal
space. It implies that a T cell–mediated response must be involved,
because cyclosporine immunosuppresses mainly, if not exclusively, by
inhibiting the calmodulin-dependent phosphatase, calcineurin in T
cells.5
That a cellular form of rejection is involved in
subretinal xenograft rejection is supported by the histology, which
usually showed an intense monocytic infiltration within and around the
transplant site.3
6
7
This cellular reaction appears to
diminish with time after rejection. There is a report of only mild
rejection of human RPE xenografts in the rabbit8
and also
evidence of tolerance of a fraction of human RPE grafts to the monkey
for as long as 6 months.7
We have also observed survival
of one transplant for more than 2 months without immunosuppression,
whereas all the other control transplants rejected within weeks after
transplantation. These differences imply that several factors are
involved in the viability of these grafts.
What is interesting is that immunosuppression can be effective at a
local level and with relatively low total concentrations of
cyclosporine. Local immunosuppression does not entirely eliminate
rejection of these xenografts, however, because virtually all disappear
with time despite immunosuppression. This may be because of either the
inability of local immunosuppression to completely stem the rejection
process or of other factors that appear to influence the survival of
these xenografts.
RPE allografts in the subretinal space seem less prone to rejection
than xenografts. In mice, RPE allografts in the subretinal space
survive longer than those placed in the conjunctiva and in addition
induce a cell-mediated suppression of delayed hypersensitivity to graft
antigen.9
There are reports that RPE allografts survive
and continue to rescue photoreceptors from degeneration in the Royal
College of Surgeons rat for relatively long times.10
11
12
On the other hand, there is evidence that such allografts in rats are
slowly rejected but in an atypical, noninflammatory
manner.13
In rabbits, RPE allografts have been reported to
survive with14
or to degenerate slowly
without15
cyclosporine immunosuppression. Small human RPE
allografts have been reported to survive when placed perifoveally, but
larger ones, especially those placed over areas where the blood–brain
barrier has been disturbed, are rejected without
immunosuppression.16
That local cyclosporine
immunosuppression could prolong the survival of RPE allografts would
seem to be a reasonable expectation, but evidence that a slow allograft
rejection in the rabbit subretinal space appears unaltered by systemic
immunosuppression17
is evidence to the contrary. Further
research will be needed to determine whether there are
cyclosporine-resistant mechanisms mediating allograft and xenograft
rejection in the subretinal space.
Such research is greatly expedited by having an in vivo monitor, which
provides a means to track the behavior and survival of these retinal
transplants, noninvasively. It would be much more difficult and
time-consuming to follow the fate of these transplants relying on
postmortem histology alone. The use of the fluorescent marker GFP
allows the same transplant to be followed over time, providing clues to
the onset and extent of the rejection process. We expect that even
greater improvements in the resolution of SLO imaging will provide even
more powerful means of following rejection and survival in the living
retina in the future.
|
Footnotes
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Supported by Grant EY 03854, National Institutes of Health (Bethesda,
Maryland); and by Research to Prevent Blindness (New York, New York).
Submitted for publication October 28, 1999; revised March 6, 2000;
accepted April 13, 2000.
Commercial relationships policy: N.
Corresponding author: Peter Gouras, Department of Ophthalmology,
Columbia University, 630 W. 168 Street, New York, NY 10032.
pg10{at}columbia.edu
|
References
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Lai, C, Doi, K, Gouras, P, Ashton, P, Tsang, S, Goff, S. (1999) Intravitreal slow release of cyclosporine prolongs survival of RPE xenografts monitored in vivo by gene transferred GFP fluorescence [ARVO Abstract] Invest Ophthalmol Vis Sci 40(4),S858Abstract nr 4521.
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Top
Abstract
Introduction
