Introduction
Duchenne muscular dystrophy (DMD) is an X-linked recessive (1), fatal disorder, caused
by mutations in the dystrophin gene, that affects approximately 1 in 3,600 to 6,000
live male births, worldwide (2-4). Deletion mutations, which are the most common cause
of DMD (5), alter the open reading frame of the dystrophin gene and usually result
in a complete absence of the protein (6;7). Dystrophin protein is an important structural
component of muscle fibers connecting their cytoplasmic cytoskeleton to the membrane-bound
dystroglycan complex (DGC) (8-11), which in turn binds to laminin in the extracellular
matrix (9-11). Lack of a functional dystrophin protein in muscle leads to destabilization
of the sarcolemmal membrane, causing muscle fiber degeneration and necrosis (12;13).
Over time, skeletal muscle tissue in DMD patients is replaced with fat and connective
tissue. Despite extensive research to date, currently there is no cure for this disease.
Gene replacement has potential utility in the treatment of single-gene disorders,
such as DMD. Dystrophin cDNA transfer using viral vectors for delivery has been extensively
tested for therapeutic efficacy in the mdx mouse, a genetic and biochemical model
of DMD (6;14). Recently, the first studies of gene replacement for muscular dystrophy
in human patients were reported (15).
Since the dystrophin gene is very large with a 11 kb of coding sequence, transfer
of the full-length dystrophin cDNA into muscles of dystrophic mice was only possible
due to the development of gutted or high capacity adenoviral (HC-Ad) vectors, which
retain no viral genes and thus have a large capacity for an inserted DNA expression
cassette (16-18). The lack of viral genes in the HC-Ad vector leads to a lower induction
of anti-vector immunity than prior generation Ad vectors and has also been shown to
facilitate prolonged dystrophin protein expression in mouse models of DMD (16). However,
in the case of a disease such as DMD, in which the endogenous therapeutic protein
can be largely absent in the host, the immunological barrier to a successful gene
transfer is not limited to the host immune reaction to vector particles, but also
to the transferred therapeutic gene product (19-21).
Previous studies found that anti-dystrophin antibodies were induced by HC-Ad vector-mediated
dystrophin cDNA delivery to muscles of adult mdx mice as early as two weeks post-gene
transfer (21). The response of the host immune system to the transferred gene reflects
the normal function of the host defense system against neo-antigens. This anti-dystrophin
immunity is detected (22-25) despite the rare dystrophin-expressing ‘revertant’ fibers
that are found in mdx muscle (19-21). However, very little is known about the anti-dystrophin
immune response raised by dystrophin-deficient recipients to dystrophin gene transfer.
Therefore, we investigated the anti-dystrophin immune response by manipulating the
immune system of adult mdx mice through a temporal removal of immune cells prior to
vector-mediated murine dystrophin cDNA delivery. We utilized complementary approaches
to temporarily remove the host immune system before gene transfer. In the first set
of experiments, a low dose of irradiation was administered to deplete only the peripheral
immune cells of adult mice and was followed by self-reconstitution of the host’s peripheral
immune cells after gene transfer. In the second set of experiments, a high dose of
irradiation was administered prior to gene transfer. After gene delivery, the host
central and peripheral immune system was reconstituted by bone marrow (BM) transfer
from a syngeneic wild-type donor; this BM should comprise cells that are fully tolerant
to dystrophin protein. By employing the 2 different doses of irradiation, we explored
the relative contributions of the peripheral and central components of the immune
response to recombinant murine dystrophin. We determined whether the returning or
new host immune cells recognized the full-length murine dystrophin protein as a self-protein.
We further explored the role of regulatory T (Treg) cells (26-30) in the peripheral
and central immune response to recombinant, murine dystrophin protein.
Results
Low dose irradiation delays or eliminates anti-dystrophin humoral response
We first examined the effect of a sub-lethal 600 rad dose of whole-body-irradiation
of 6-week-old mdx mice intended to temporarily remove peripheral immune cells. Immune
responses against HC-Ad vector-mediated murine dystrophin expression in dystrophic
muscles was studied longitudinally. A control group was not irradiated prior to gene
delivery. Within 24 hours post-irradiation mice in both groups each received an intramuscular
injection of HC-Ad vector carrying the murine dystrophin cDNA driven by the MCK promoter
(1.5-2.0 × 1010 genome copies in each tibialis anterior (TA) muscle). Treated and
control mice were analyzed at 4, 8, and 12 weeks after irradiation and gene delivery.
At 4 weeks post-gene transfer, mdx mice that had been irradiated prior to vector injection
had no detectable anti-dystrophin humoral response (Fig. 1A, wk4), whereas control
mice demonstrated an anti-dystrophin humoral response at 4 weeks post-treatment (Fig.
1A, wk4). At 8 weeks post-vector transfer the irradiated mdx mice demonstrated a variable
level of anti-dystrophin humoral immunity. Three of the 5 mice showed a dystrophin-specific
humoral response and two mice showed no response to dystrophin protein. The level
of the response in mice that did produce anti-dystrophin antibodies was lower compared
to control mice, all of which demonstrated a dystrophin-specific humoral response
(Fig. 1A, wk8). A third group of mice was analyzed at 12 weeks post-vector injection.
One of 4 irradiated mice produced an anti-dystrophin humoral response, while the other
3 mice did not (Fig. 1A, wk12). All control mice showed a dystrophin-specific humoral
response at 12 weeks (Fig. 1A and Table 1).
HC-Ad vector-mediated dystrophin expression in low-dose irradiated mdx muscles
Dystrophin expression in the vector-injected muscles of mdx mice was analyzed by immuno-staining
of muscle cryo-sections. Dystrophin-expressing fibers were scattered throughout the
vector-injected muscle tissue of irradiated mice at 4, 8, and 12 weeks post-treatment.
In contrast, control mice did not show muscle dystrophin expression above the background
level of revertant fibers observed in untreated muscle at any time-point (Fig. 1B
and Table 1). The number of dystrophin-expressing muscle fibers in the irradiated,
vector-treated mice decreased with time correlating temporally with the production
of anti-dystrophin antibodies (Fig. 1B). The reduction in the number of dystrophin-expressing
fibers over time was observed even in mice that did not produce anti-dystrophin antibody,
suggesting that the humoral response was not solely responsible for the decrease in
dystrophin protein expression.
Lymphocytes in draining lymph nodes and vector-injected muscle after low-dose irradiation
Since antibody production and B cell population are directly related to each other,
B cell levels in the lymph nodes draining the vector-injected TA muscles were analyzed
to ensure that the peripheral B cells in the irradiated mice had returned to levels
comparable to untreated mdx mice. At all 3 time-points, the level of B cells, which
were identified by CD19 expression, in draining lymph nodes of vector-treated mice
was not significantly different from untreated mdx mice suggesting a return to the
full complement of B cells after irradiation (Fig. 2A). The number of B cells in the
irradiated, vector-treated mice, however, increased gradually from week 4 to week
12. The increase in the number of B cells coincided with the development of anti-dystrophin
antibodies suggesting that the self-reconstituted B cells eventually recognized dystrophin
protein as a neo-antigen and generated an anti-dystrophin immune response. At 4 weeks
post-treatment, the number of B cells in the draining lymph nodes of the control mice
was significantly higher than both the irradiated, vector-injected and the untreated
groups (Fig. 2A), suggesting an early vector-mediated B cell expansion that correlated
with the high level of anti-dystrophin humoral response observed in control mice.
We also investigated the infiltration of CD4+ and CD8+ T cells in vector-injected
muscles of mdx mice to assess the cellular immune reaction following murine dystrophin
expression. Since muscle infiltration of CD4+ and CD8+ T cells is a disease-associated
phenotype of mdx mouse muscle and is thought to promote muscle fiber necrosis (31),
infiltrating T cells are seen in dystrophic mdx muscles with and without dystrophin
gene transfer. We compared the number of T cells infiltrating vector-injected muscle,
with or without irradiation, to age-matched untreated muscle.
Levels of infiltrating CD4+ and CD8+ T cells in muscles of low-dose irradiated, vector-treated
or control mice were not significantly different from untreated mice at week 4 post-treatment
(Fig. 2B and 2C). At week 8 CD4+ and CD8+ T cells increased in the vector-injected
TA muscles of both irradiated and non-irradiated hosts compared to untreated muscle,
indicating the development of a T cell-mediated response following dystrophin vector
expression that was not affected by irradiation (Fig. 2B and 2C). By week 12, levels
of CD4+ and CD8+ T cells had increased in all groups to similar levels (Fig. 2B and
2C).
The pattern of T cell infiltration for both CD4+ and CD8+ T cells observed on muscle
sections was consistently different in the vector-treated, irradiated mice compared
to the control group (Fig. 3). At week 4, T cells were scattered throughout the muscle
tissue in irradiated, vector-injected mdx muscles, similar to those in age-matched
untreated mdx muscles. In the control group, however, in addition to scattered infiltrating
cells, large numbers of cells were seen to cluster around one or multiple adjacent
fibers, and groups of necrotic muscle fibers were seen as early as 4 weeks post-treatment.
Therefore, it appeared that the transduced muscle fibers were targeted by a specific
cell-mediated host immune response in the absence of irradiation prior to gene transfer.
In the irradiated group a similar pattern was only seen in the vector-injected muscles
at week 12 post-treatment (data not shown).
High-dose irradiation delays anti-dystrophin humoral response
We next examined the effect of removing central and peripheral immune cells of mdx
mice by 900 rads irradiation prior to intramuscular dystrophin gene delivery (1.5×1010
genome copies in each TA muscle). The immune systems of irradiated mice were then
reconstituted by transfer of wild-type BM from C57BL/10 (B10) donors. Mice were analyzed
at 4, 8, or 12 weeks post-treatment. We had two control groups: one that underwent
only gene delivery without receiving other treatments and one that was irradiated
and received adoptive transfer of B10 BM without gene transfer (Table 1).
We considered what would be an expected time course to clear the initial load of vector
capsid proteins. To explore this experimentally, C2C12 myoblast cells were infected
in vitro with Ad vector. Harvested cell samples at various time points for 48 hours
were assayed for Ad vector capsid proteins by Western blot. Nearly all Ad capsid proteins
were degraded in vitro by 12 to 24 hours post-infection (data not shown). Despite
likely differences between viral protein degradation in vitro and in vivo, the in
vitro results gave us an approximate time point for progressing to our in vivo studies.
Balancing the desire to maximize the time for viral proteins to degrade prior to BM
transfer with a need to minimize mouse loss due to high dose irradiation, we performed
BM transfer at about 15 hours post-gene transfer. BM transfer consisted of tail vein
intravascular (IV) injections of 4.0-5.0 × 106 B10 BM cells.
At 4 weeks post-gene transfer none of the mice that had been irradiated and received
B10 BM produced anti-dystrophin antibodies. However, the vector-injected mice that
had not been irradiated produced anti-dystrophin antibodies at this time point, similar
to the control group in the low-dose irradiation study (Fig. 4A). Unlike the low-dose
study group, in which a few mice produced anti-dystrophin antibodies at week 8 post-treatment,
at this time-point none of the irradiated, vector-injected mice that received B10
BM produced an anti-dystrophin humoral response (Fig. 4A). By 12 weeks post-treatment,
however, we observed anti-dystrophin humoral response in all of these mice (Fig. 4A).
HC-Ad vector-mediated dystrophin expression in high-dose irradiated mdx muscles
Dystrophin protein expression in the muscles of treated mdx mice was assessed by immunohistological
assay, as before. In the high-dose irradiation group we first determined that adoptive
transfer of B10 BM alone did not give rise to dystrophin-expressing fibers above the
level in untreated age-matched mdx muscles, for at least 12 weeks post-treatment (Fig.
4B). At all time-points post-treatment, dystrophin expression was significantly higher
in the muscles of the irradiated mice that had received vector and B10 BM compared
to the control groups (Fig. 4B, wk4). Compared to week 4, at week 8 post-treatment,
the level of dystrophin expression increased in B10 BM recipient mice that received
dystrophin vector (Fig. 4B; wk8). The subsequent decline in dystrophin expression
level at week 12 post-treatment correlated with the production of anti-dystrophin
antibodies by these mice.
Lymphocytes in the host lymph nodes and vector-injected muscles after high-dose irradiation
and BM transfer
The lymph nodes draining vector-treated muscles of the high-dose irradiated mice were
analyzed to examine peripheral lymphocyte levels. The percentage of B cells in the
draining lymph nodes was similar among all groups at 4 and 8 weeks post-treatment
(Fig. 5A). By week 12 post-treatment the B cell levels in all groups had decreased
(Fig. 5A, wk12).
We also looked at T cell infiltration in the treated muscles at the same time points.
At week 4 post-treatment, the number of CD4+ T cells was significantly lower in the
muscles of the irradiated mice that received dystrophin vector and B10 BM compared
to the non-irradiated, vector-injected mice (Fig. 5B and 5C). CD8+ T cells were at
comparable levels in the irradiated and non-irradiated, vector-injected mice. Starting
at week 8, however, muscle infiltrating CD4+ and CD8+ T cells increased in the irradiated,
vector-injected mice at comparable levels to the non-irradiated, vector-injected mice
(Fig. 5B and 5C). The levels of infiltrating CD4+ and CD8+ T cells remained stable
at week 12 post-treatment compared to week 8 in the irradiated, vector-injected group
that received B10 BM (Fig. 5B and 5C).
Infiltrating T cells were observed scattered throughout in the high-dose irradiated,
vector-injected muscles at all time points (data not shown) in contrast to clustering
around specific muscle fibers in non-irradiated, vector-injected muscle (Fig. 3).
These observed differences in the pattern of muscle T cell infiltration was similar
to that observed in the low-dose irradiation study.
Regulatory T cells in treated muscles
An important cell population in modulating immunity is a regulatory subset of CD4+
T cells expressing the fork head box 3 (Foxp3) transcription factor, commonly referred
to as Foxp3+ regulatory T (Treg) cells (30;32;33). We compared the infiltration of
Treg cells in treated muscles at each time point to explore a possible role of these
cells in modulating the immunity that we observed in treated mdx mice. In the low-dose
irradiation study, the number of muscle-infiltrating Treg cells was comparable at
each time point for the vector-treated groups with and without irradiation (Fig. 6A).
In our treatment groups there was a decrease from week 4 to week 8 followed by an
increase from week 8 to week 12, a pattern that was also observed in untreated mdx
mouse muscle, suggesting a contribution of the dystrophic disease progression in muscle.
In the high-dose irradiation study there was a significant increase in Treg cell infiltration
starting at week 8 post-treatment in the irradiated, vector-injected mice, but not
in the non-irradiated, vector-injected mice (Fig. 6B). The number of these cells further
increased at week 12 and remained significantly higher in the irradiated, vector-injected
B10 BM recipients as compared to the non-irradiated, vector-injected mice (Fig. 6B).
We also examined the level of muscle infiltration of programmed death-1 (PD-1) expressing
cells, that have been shown to have immune regulatory effects (34-36). There was no
significant difference in the number of PD-1+ muscle-infiltrating cells in different
treatment groups at different time points in mice receiving either low-dose or high-dose
irradiation (data not shown).
Discussion
In studies on therapeutic gene replacement for DMD, the immune response of the dystrophic
host to vector-mediated dystrophin protein expression is an important limiting factor
with the potential to cause rejection of recombinant dystrophin. Further understanding
of the immunological reactions against dystrophin, immune mechanisms that may modulate
anti-dystrophin immunity, and manipulations that may reduce anti-dystrophin immune
responses in a dystrophic host provides the potential to develop approaches designed
to prolong dystrophin expression in the recipient of therapeutic gene transfer.
A temporal analysis of the effect of irradiation alone or irradiation combined with
BM transfer on the immunity against vector-mediated full-length dystrophin expression
in mdx muscle has not been previously reported. Previous studies have examined the
effects on dystrophic muscle of adoptive transfer of wild-type BM to irradiated mdx
mice (37), muscle precursor cells to irradiated mdx mice (38), or BM-derived stem
cells to non-irradiated mdx mice (39). In the current study we limited the dose of
irradiation to a maximum of 900 rads, as in other studies (40), to avoid damage to
muscle stem cells (41). The use of the HC-Ad vector for gene delivery allowed exposure
to all epitopes of an expressed full-length murine dystrophin cDNA.
In our studies, BM transfer alone to mdx mice did not increase the level of muscle
dystrophin expression over the background level in untreated mdx mice at any time-point,
which confirmed the findings of others (37;42). Therefore, the dystrophin expression
observed after dystrophin vector delivery following irradiation and BM transfer is
vector-mediated.
Our findings demonstrate that irradiation of the dystrophic mdx host prior to intramuscular
vector injection led to a delayed, reduced, or absent humoral response against dystrophin
protein. The changes in anti-dystrophin immunity in treated mice allowed us to study
the progression of host immunity against dystrophin and its effect on dystrophin expression
over a period of 12 weeks. The observation that both low-dose and high-dose irradiation
delayed the development of an anti-dystrophin antibody response is significant compared
to the previous findings of more rapid host immunity against dystrophin (21).
The delay in development of an anti-dystrophin humoral response was greater in the
high-dose irradiated group compared to the low-dose irradiated group, as no anti-dystrophin
humoral response was observed at 8 weeks post-treatment. Our results suggest that
the more complete immune cell depletion (both central and peripheral immune cells)
and reconstitution with dystrophin-tolerant immune cells results in greater suppression
of anti-dystrophin immunity induced by dystrophin gene transfer to the dystrophin-deficient
mdx host.
The level of CD4+ and CD8+ T cell infiltration in untreated dystrophic mdx muscle
(31;43) and in dystrophin vector-injected mdx muscle (19;44) has been previously investigated.
The dystrophic pathology of mdx mouse muscle includes a high level of T cell infiltration
in the damaged tissue that is thought to promote the pathology associated with the
disease (31;43). It has been shown that specific immune modulations that lead to absent
or reduced T cells in the mdx mouse can ameliorate the disease process in muscle (31;43;45)
and, in a gene transfer setting, reduce immunity against dystrophin protein in the
host (18;44). Our present study compared the levels of infiltrating CD4+ and CD8+
T cells in vector-injected muscles to untreated mdx mouse muscle at different time
points. Thus, we evaluated the role that these T cell sub-populations may play in
a gene therapy setting as both disease and the immunity against recombinant dystrophin
progressed. In contrast to the observed correlation with the anti-dystrophin humoral
response, irradiation prior to gene transfer does not seem to affect levels of infiltrating
T cells in vector-injected muscles of the mdx host during 12 weeks post-treatment
in either low- or high-dose irradiation groups. Interestingly, however, the pattern
of T cell infiltration in muscle tissue did appear to be affected by irradiation.
In the early stages after gene transfer, the finding of muscle fibers surrounded and
invaded by T cells was only observed in the non-irradiated, vector-injected muscles.
This focal pattern of T cell infiltration was not observed in the irradiated vector-injected
and untreated mdx mice at this time point. Double-staining of T cells and dystrophin
in the attacked fibers was not feasible because only necrotic fiber remnants remained.
Nonetheless, the distinctive pattern of T cell infiltration plus the absence of dystrophin-expressing
fibers in non-irradiated, vector-injected muscles at early stages post-gene transfer
suggested that transduced fibers were targeted for immune attack. The same pattern
of T cell infiltration has also been observed in previous studies in which transfer
of muscle precursor cells from dystrophin-normal donors to irradiated mdx mice, that
had received mixed BM from muscle cell-donors and mdx donors, resulted in infiltration
of CD4+ and CD8+ T cells around dystrophin-expressing muscle fibers at 3-4 months
post-treatment (46).
Overall, our analysis of T cell infiltration in treated mdx muscle indicates that
even though CD4+ and CD8+ T cells infiltrates are found in irradiated, vector-treated
mdx muscles, this host immune response does not lead to an immediate rejection of
dystrophin protein in the vector-injected muscles of irradiated mice for at least
12 weeks post-treatment. In contrast, T cell infiltration in the vector-injected muscles
of non-irradiated mice appeared to reject dystrophin-expressing fibers, suggested
by the pattern of T cell infiltration surrounding muscle fibers and the absence of
dystrophin expression as early as 4 weeks post-treatment. One limitation of the analysis
of cellular immunity observed in treated or untreated mdx muscle was that the antigens
inducing immunity cannot be specifically determined.
To decipher the mechanism underlying the delayed and diminished host immune response
observed in both low- and high-dose irradiated groups, we examined infiltration of
Treg cells and PD-1+ T cells in treated and control mdx muscles. Only Treg cells were
different among the groups. We have previously observed Foxp3+ Treg cells to be at
slightly higher levels in untreated mdx mouse muscle compared to age-matched B10 muscle
(unpublished data). In this study, we observed even higher levels of Treg cells in
vector-injected muscle of mdx mice 8 and 12 weeks after receiving high-dose irradiation
and reconstitution of the immune system with B10 BM. This increase in Treg cells suggested
their role in the diminished anti-dystrophin immune response observed in the setting
of central and peripheral immune cell depletion followed by reconstitution. In contrast,
Treg cells did not appear to play a role in the delayed immunity associated with depletion
of the peripheral immune cells alone followed by self-reconstitution (low-dose irradiation).
Taken together, these results suggest a role for Treg cells in the suppression of
immunity induced by dystrophin vector gene transfer followed by reconstitution of
the central immune system. The adoptive transfer of whole BM from B10 mice may have
contributed to the relatively high level of Treg cells in treated muscles since the
transferred BM is likely to contain cells tolerant to B10 self-antigens, including
muscle proteins. The exact role of Treg cells in the setting of dystrophin gene delivery
to dystrophic muscle will require further study.
In summary, we show that manipulation of the host immune system by irradiation of
the adult dystrophic mdx mouse prior to intramuscular HC-Ad dystrophin vector delivery
results in a delayed and diminished humoral immune response to vector-mediated dystrophin
protein expression in the adult mdx mouse. The delay in the response correlated with
a significantly slower rate of elimination of vector-mediated dystrophin protein expression
in treated mdx muscle compared to muscle of non-irradiated vector-injected mice. Although
there are technical limitations to determining the antigen specificity of T cells
infiltrating muscle, the pattern of T cells surrounding and invading muscle fibers
suggests that viral vector-mediated dystrophin transduction contributes to the antigenic
targets, particularly since this pattern of T cell infiltration is diminished and
delayed by irradiation. The failure to eliminate anti-dystrophin immunity with the
central and peripheral immune depletion achieved by irradiation supports the contention
that dystrophin expression in dystrophin-deficient, dystrophic muscle is immune-stimulatory,
a finding which has significant ramifications for dystrophin gene replacement therapy.
Materials and Methods
Mice
Wild type (C57BL/10J) and mdx (C57BL/10ScSnDmdmdx/J) mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). All mouse studies were performed under approved protocols
by the University of Pittsburgh Institutional Animal Care and Use Committee.
Dystrophin high-capacity adenoviral vector
The HC-Ad vector contains a full-length murine dystrophin cDNA under the control of
the muscle creatine kinase (MCK) promoter, the left and right viral inverted terminal
repeats (ITRs) of adenovirus serotype 5 and hypoxanthine-guanine phosphoribosyl transferase
(HPRT) ‘stuffer’ DNA. The construction of this vector has been fully described previously
(18;47).
Mouse irradiation
Mice were irradiated at the age of 5-6 weeks in a LINAC irradiator at either a low
dose (600 rads) or a high dose (900 rads). Specialized care included autoclaved cages,
autoclaved food, and acidified water (pH 2.6) from the day before irradiation until
the end of each study. In the low-dose irradiated group the immune system was allowed
to spontaneously reconstitute following gene transfer and in the high-dose irradiated
group the mice received whole BM from wild-type C57BL/10 mice at 12 hours post-gene
transfer. BM was transferred by intravascular (IV) injections of 4.0-5.0 × 106 cells.
Intramuscular vector injections
Irradiated and non-irradiated mice received inhalational anesthesia with isoflurane
and were injected with 1.0-2.0 ×1010 genome copies of HC-AdmDYS vector intramuscularly
in the TA muscle bilaterally. Each muscle was injected with a volume of 20μl in PBS,
using a 28G needle (B-D; Franklin Lakes, NJ).
Total muscle protein extract (TMPE) preparation
Freshly isolated mouse muscle was homogenized in TEES buffer (50mM Tris-HCl pH 8.0,
5mM EGTA pH 7.4, 5mM EDTA pH 8.0, 5% SDS) and incubated on ice for 45 minutes. Samples
were then sonicated briefly and centrifuged at 14,000 rpm for 20 minutes at 4°C. Supernatant
was collected and stored at −80°C.
Serum preparation
Blood was collected by cardiac puncture shortly after sacrifice and centrifuged at
10,000 rpm for 15 minutes at 4°C. Supernatant was collected and stored at 4°C.
Gel electrophoresis and Western blotting
The membranes with immobilized murine dystrophin protein used for the assay were generated
from wild-type C57BL/10J (B10) total muscle protein extracts (TMPE) electrophoresed
on SDS-PAGE. In brief, TMPE from B10 mice were run on 5% Acrylamide gel (Bio-Rad;
Hercules, CA) for 3 hours at 110V. Protein samples were transferred from the gel to
nitrocellulose membrane (Amersham biosciences) for 1.5 hours at 110V at 4°C. The membrane
was blocked in 5% milk/1% sheep serum/TBST (10mM Tris pH 8.0, 0.15M NaCl, 0.5mM Tween-20)
overnight at 4°C. The membrane was then cut in pieces with a dystrophin protein band
in each piece. The pieces of membrane were then each incubated with a diluted serum
sample (1:300) in TBST for 1.5 hours, then with HRP-conjugated sheep anti-mouse IgG
(GE Healthcare; Piscataway, NJ) diluted in TBST for 45 minutes at room temperature.
ECL detection reagent (GE Healthcare; Piscataway, NJ) was used to detect the chemiluminescent
signal and Kodak film was used for visualizing the signal. Presence or absence of
a dystrophin band was recorded. The initial exposure time was 15-30 minutes and to
confirm the absence of a band in irradiated mice an additional film was exposed to
the membranes overnight.
Bone marrow (BM) preparation
Tibia and femur of adult wild-type mice were dissected with complete removal of surrounding
muscle. Syringes with 26G needles were used to flush the BM out of the bones onto
a cell strainer using RPMI-1460 medium supplemented with 2% fetal bovine serum (FBS),
5% penicillin/streptomycin, and 10U/ml heparin (Sigma-Aldrich; St. Louis, MO). BM
was then washed with a serum-free RPMI-1460 medium (5% penicillin/streptomycin, 20mM
HEPES) and counted. For adoptive transfer, cells were re-suspended in serum-free medium
at a concentration of 2.0×107 cells/ml. Cell suspension (200 μl) was injected I.V.
(4-5×106 cells/mouse) using a 28G needle (B-D).
Flow cytometry
Single-cell suspensions were prepared from lymph nodes by mechanical disaggregation
through a 40μm cell strainer (Fisher) into PBS. Cells were incubated with rat anti-CD4
(FITC), CD25 (APC), and Foxp3 (PE) (ebiosciences; San Diego, CA; 11-0042-82, ,17-0251-81,
12-5773-80, respectively) and rat anti-CD8 (PE), CD19 (FITC), and CD11c (FITC) (Pharmingen;
San Jose, CA; 553032, 557398, and 557400, respectively) in FACS staining buffer (ebiosciences;
San Diego, CA) as indicated by the manufacturer’s instructions. The stained cells
were then analyzed by a B-D SLR II flow cytometer.
Muscle Tissue Processing
Freshly dissected muscle was incubated in 2% paraformaldehyde/PBS on ice for 2 hours,
then transferred into 30% sucrose overnight at 4°C. The next day the muscle tissue
was snap-frozen in 2-methylbutane cooled with dry ice and stored at −80°C.
Immunohistochemistry of inflammatory cells
10μm cryo-sections of muscle samples were prepared. Cross-sections (4-5 sections per
TA per mouse) were made from different parts of each TA muscle for this staining.
Sections were rehydrated in PBS, blocked in peroxidase blocking reagent (DAKO Cytomation;
Carpinteria, CA) for 5 minutes, and then blocked in 10% goat serum/PBS for 1 hour
at room temperature. The primary antibody incubations using rat anti-CD4, Foxp3, and
PD-1 (ebiosciences; San Diego, CA; 16-0041-81, 14-4771-80, and 13-9985-81, respectively)
and rat anti-CD8 (Pharmingen; San Jose, CA) purified antibodies diluted in 10% goat
serum/PBS were done for 1.5 hours at room temperature. Sections (except for PD-1 sections
because PD-1 primary antibody was biotinylated) were incubated for 1 hour with secondary
biotinylated goat anti-rat IgG (Pharmingen; San Jose, CA) diluted in DAKO antibody
diluent (DAKO Cytomation; Carpinteria, CA). Sections were incubated in ABC Vectastain
avidin-HRP detection solution (Vector Laboratories; Burlingame, CA) for 30 minutes
at room temperature and DAB peroxidase substrate solution (Vector Laboratories; Burlingame,
CA) for 4 minutes. Eosin counterstaining was performed for visualization of muscle
fibers. To analyze infiltrating cells in each group, the total number of cells per
cross-section of vector-injected TA muscles was counted and the average number of
cells from sections from different mice was calculated.
Immuno-fluorescence detection of dystrophin
10μm cryo-sections, as mentioned above, were rehydrated with PBS, blocked first with
avidin and biotin block (Vector Laboratories; Burlingame, CA), and then with mouse
IgG block (Vector Laboratories; Burlingame, CA), according to the manufacturer’s instructions.
Incubation with primary anti-DYS was done for 3 hours. Sections were then incubated
with biotinylated goat anti-mouse IgG (Pharmingen; San Jose, CA) and tertiary FITC-conjugated
donkey anti-goat IgG.
Statistical Analysis
The statistical analysis was performed by student’s t-test, in which a treatment group
and a control group, or two treatment groups were compared as unpaired sets. Values
of variables were presented as the mean with standard deviation (SD). In all experiments,
P values <0.05 were considered significant.