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Blood First Edition Paper, prepublished online November 17, 2009; DOI 10.1182/blood-2009-08-239509
Induction of immune tolerance to FIX by intramuscular AAV gene transfer is
independent of the activation status of dendritic cells
Running Head: DC activation and FIX tolerance
Arpita S. Bharadwaj,1 Meagan Kelly,1 Dongsoo Kim1 & Hengjun Chao1
1
Division of Hematology/Oncology, Department of Medicine,
Cancer Institute, Immunology Institute
Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029
Correspondence: Dr. Hengjun Chao
Division of Hematology/Oncology, Box 1079
Mount Sinai School of Medicine
One Gustave L. Levy Place, New York, NY 10029
Phone: 212-241-9567, Fax: 212-426-4390, Email: [email protected]
Abbreviations: AAV, adeno-associated virus. DC, dendritic cells. FIX, coagulation factor
IX. hFIX, human FIX.
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Copyright © 2009 American Society of Hematology
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Abstract
The nature of viral vectors is suggested to be a significant contributor to
undesirable immune responses subsequent to gene transfer. Such viral vectors
recognized as “danger signals” by the host immune system, activate dendritic cells (DCs),
causing unwanted anti-vector and/or transgene product immunity. We recently reported
efficient induction of immune tolerance to FIX by direct intramuscular injection of
AAV1-FIX. AAV vectors are non-pathogenic and elicit minimal inflammatory response.
We hypothesized that the non-pathogenic nature of AAV plays a critical role in induction
of tolerance following AAV gene transfer. We observed inefficient recruitment and
activation of DCs subsequent to intramuscular injection of AAV. To further validate our
hypothesis, we examined immune responses to FIX following intramuscular injection of
AAV with simultaneous activation of DCs. We were able to achieve phenotypic and
functional activation of DCs following administration of LPS and anti-CD40 antibody.
However, we observed efficient induction of FIX tolerance irrespective of DC activation
in mice with different genetic and MHC backgrounds. Furthermore, activation of DCs
did not exaggerate the immune response induced following intramuscular injection of
AAV2. Our results demonstrate that induction of FIX tolerance following AAV gene
transfer is independent of DC activation status.
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Introduction
Gene therapy is emerging as a valuable alternative treatment for human diseases.
However, adverse immune responses subsequent to gene transfer, such as severe
cytotoxic T lymphocyte response (CTL) and formation of inhibitory antibodies against
transgene products,1-4 need to be addressed for successful application of gene therapy in
human patients. The primary step would be to determine critical factors that may decide
or control the ultimate immunological outcomes in gene transfer. This will provide a
fundamental insight into the mechanism accounting for the immune responses subsequent
to gene transfer, leading to a better understanding and then final resolution of the adverse
immune responses.
A variety of inherent factors, in conjunction with gene transfer, can be
encountered and recognized as a class of “danger signals” by the host immune system.
2,5
Such danger signals elicit the innate immunity of the host, thus stimulating and causing
the maturation and activation of quiescent antigen presenting cells (APC). 2,5,6 The
activated APCs in turn present the processed antigen with appropriate MHC molecules to
antigen-specific T cells, to initiate the relevant immune response. 2,5 Dendritic cells (DC)
are a major type of professional APCs, which act as the central decision-maker of the
immune system.6-8 Activation of dendritic cells by the danger signal is a critical step in
deciding the ultimate immunological outcome. 5,6,8,9 Quiescent (immature or mature) DCs
are considered tolerogenic and capable of inducing T cell deletion, anergy or regulatory T
cells. Whether a T cell is tolerized or activated to become an effector cell, depends on
the activation status of the antigen presenting cells. The mature and activated DCs initiate
priming of the antigen-specific CD4+ helper T cells, leading to immune responses to
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relevant targets such as the delivery vector.6,9,10 Not all transgene products are
immunogenic, therefore not recognized as “danger” by the host immune system.
However, the anti-viral vector immunity can instigate collateral adverse immune
responses against the transgene product.2 If the transgene product itself is immunogenic,
the anti-viral vector immunity can worsen the intensity of the undesirable immune
responses against the transgene product.
A critical factor in relation to gene transfer that can be identified as a “danger
signal” by the host organisms is the nature of the viral gene delivery vector. Many gene
transfer vectors are recombinant derivatives of viruses, such as adenovirus and retrovirus,
the majority of which are pathogenic and immunotoxic.1,2 Although the final recombinant
viral vectors that are used in gene delivery are devoid of potential pathogenic viral
component, vector-related immunotoxic incidents have been observed in many gene
transfer studies.1 Among all viral vectors tested in gene therapy studies, adeno-associated
virus (AAV) is the only virus that is not associated with any known human disease. The
non-pathogenic nature of AAV does not present itself as a “ danger signal” to the host. It
therefore causes only a minimum level of vector-related toxicity and immune responses
in AAV-based gene transfers.2,11,12 This makes AAV an attractive gene transfer vector
compared with other gene transfer vectors derived from pathogenic viruses.
FIX gene transfer for hemophilia B treatment is a good model for gene therapy
studies. Multiple strategies have been used for FIX gene transfer including the use of
viral vectors. AAV has been extensively tested for hemophilia B gene therapy. Direct
intramuscular injection of AAV has been shown to be a convenient, safe and potentially
effective approach for FIX gene transfer.13-15 Intramuscular injection of AAV does not
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cause severe cellular immune response such as CTL in rodents, canine, or human
patients. Such severe immune responses were however substantial following gene
transfers using recombinant adenoviral vector.16 Formation of inhibitory anti-FIX
antibodies, which is the major complication in FIX replacement to treat hemophilia B,
has also been observed in pre-clinical studies of intramuscular AAV gene transfer.13,17-19
Efficient induction of immune tolerance to FIX is critical for the success of
hemophilia treatment. Many factors have been proposed to contribute to induction of
immune tolerance or immunity to FIX following muscular AAV gene transfer.13,14,17-19 It
is however inconclusive and still an ongoing debate as to the immunological consequence
and the significance of these factors. It needs further investigation.
We recently reported, efficient induction of immune tolerance to FIX by direct
intramuscular injection of AAV serotype one vector (AAV1) in mice with diverse genetic
and immunological backgrounds.13-15 AAV has proven to be much less immunogenic in
contrast to other pathogenic viral vectors such as recombinant adenoviral vectors.11,12 It is
conceivable that lower incidence of immunity against transgene product in AAV gene
transfer may be ascribed to the non-pathogenicity of AAV and the consequent
inefficiency of activated DC to stimulate an immune response. It was also reported that
excess local immunity at the injection site could increase anti-FIX immunity in the
context of intramuscular AAV gene transfer.20 We therefore hypothesized that the nonpathogenic property of the AAV vector is one of the major factors that facilitates
induction of immune tolerance to FIX following intramuscular AAV1-FIX gene transfer.
In the current study, we first examined the status of DCs in the context of
intramuscular AAV gene transfer. We found that the DCs remained in a steady state
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following intramuscular injection of either AAV1 or AAV2. We then investigated
immune responses to FIX following intramuscular injection of AAV1 concomitant with
full activation of DCs by LPS and anti-CD40 antibody. Our data demonstrated that
despite activation of DCs, no change in the ultimate outcome of immune tolerance
induced by intramuscular injection of AAV1 vectors was observed. We accordingly
concluded that, induction of FIX tolerance by intramuscular AAV gene transfer is
independent of the activation status of dendritic cells.
Material and Methods
AAV vector production, animal care and procedures
The AAV-hFIX vectors were made using a three plasmid transfection scheme as
previously described.13,15 C57BL/6, Balb/c and C3H mice were purchased from Jackson
Laboratory (Bar Harbor, Maine, USA). All the mice were maintained in pathogen-free
animal facilities at Mount Sinai School of Medicine, and treated in accordance with the
guidelines of the Institutional Animal Care and Use Committee of Mount Sinai School of
Medicine, which approved this study. AAV injections, animal procedures and plasma
sample collection were conducted as previously described.13,15 For in vivo dendritic cell
activation, eight to ten week old mice were injected with 100 μg/mouse of anti-CD40
antibody (Rat anti-mouse CD40, IgG2a, Clone FGK45, Axxora, LLC. San Diego, CA) in
the footpad and 100 μg/mouse LPS (Lipopolysaccharide, Sigma Aldrich, St. louis, MO)
via intraperitoneal injection or anti-CD40 antibody alone on day 0. The draining lymph
nodes and spleens were collected 6, 24, 72 and 120 hours after AAV and anti-CD40
antibody±LPS injection for characterization of dendritic cells. Two units of recombinant
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hFIX (rhFIX; Genetics Institute, Cambridge, MA) emulsified in 100 μl of complete
Freund’s adjuvant (CFA; Pierce Biotechnology, Rockford, IL) was injected
subcutaneously for boosting the immune response to hFIX or for verification of hFIX
immune tolerance.
Detection of hFIX antigen and anti-hFIX antibodies
Human FIX antigen was measured by an enzyme-linked immunosorbent assay
(ELISA) as previously described.13 Anti-hFIX antibody-specific ELISA was performed
to detect anti-hFIX antibodies as previously described.13,15
Phenotypic characterization of DCs
Single cell suspensions were prepared by mechanical disintegration of the isolated
draining lymph nodes and spleen, followed by red blood cell lysis (BD Pharmingen, CA).
The cells were suspended in 100 μl of FACS staining buffer (5 ml PBS, 100 μl each of
normal mouse, rabbit and human serum, 333 μl 30% BSA, 5 ml HBSS complete with
BSA and 100mM EDTA), and then stained with the following florochrome conjugated
antibodies CD11c-PE (1:200), CD80-APC (1:200), CD86-APC (1:200) (BD Pharmingen,
CA) and MHCII-Biotin (eBiosciences, CA). The cells were incubated with the antibody
mixture for 30 minutes on ice and washed twice by the addition of 1ml FACS buffer.
Cells that received biotin conjugated antibodies were incubated an additional 30 minutes
with avidin-APC-Cy7 (BD Pharmingen, CA). The cells were then washed twice and
stored in 1x formalin buffer prior to analysis. The cells were read on BD LSRII flow
cytometer equipped with the BD FACSDiva software (Becton Dickinson, San Jose, CA).
The data was analyzed using FlowJo analysis software (TreeStar, OR).
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Cytokine Profile
Splenocytes were suspended in complete RPMI-1640 and cultured for 4 hours at
37°C in a CO2 incubator. GolgiSTOP (4µl/6ml culture,BD Pharmingen, CA) was added
and incubated for an additional 4 hours at 37°C. The cells were collected and stained for
surface antigen CD11c-PE (1:200) (BD Pharmingen, CA). The cells were then fixed and
permeabilized as per manufacturers recommendation (BD Cytofix/Cytoperm Plus
Fixation/Permeabilization kit, BD Pharmingen, CA). The cells were subsequently stained
for intracellular cytokines IL-12-APC, IL-6-PE and IL-10-FITC (BD Pharmingen, CA).
The cells were evaluated on BD LSRII flow cytometer equipped with the BD FACSDiva
software (Becton Dickinson, San Jose, CA). The data was analyzed using FlowJo
analysis software (TreeStar, OR).
Statistical analysis
The data was analyzed using GraphPad Prism (GraphPad Software, CA).
Statistical differences between the various experimental groups were evaluated by twotailed, unpaired T test. p<0.05 was considered statistically significant.
Results
In the current study we proposed to examine the role of DCs in AAV1 induced
immune tolerance to FIX. For a sensitive as well as dependable evaluation of DC
motivation, recruitment and activation following intramuscular injection of AAV, we
performed preliminary experiments to determine the appropriate duration, route and
mode of DC activation. We compared different agents that are known and defined for DC
activation, such as LPS (Lipopolysaccharide) and anti-CD40 antibody administered either
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subcutaneously (footpad) or intraperitoneally. 21-23 We examined number of CD11c+
cells as well as expression of MHCII and co-stimulatory molecules (CD80 and CD86) on
CD11c+ cells in draining lymph nodes (Figure 1A) and spleen (Figure 1B) of the mice at
6, 24, 72 and 120 hours after administration of anti-CD40 antibody alone or LPS plus
anti-CD40 antibody or PBS. We observed that the combination of LPS and anti-CD40
antibody was optimal for the activation of DCs (Figure 1A-B). We also observed that
DC activation peaked at 24 hours upon administration of the DC stimulator via the
subcutaneous (footpad) route (Figure 1A-B). We thus decided to evaluate the status of
DCs at 24 hours following intramuscular injection of AAV and/or administration of antiCD40 antibody together with LPS.
Direct intramuscular injection of AAV vectors does not motivate or activate DCs
We first examined the numbers and activation status of DCs subsequent to
intramuscular injection of AAV vectors. Cohorts of eight to ten week old C57BL/6 mice
(n=4 per cohort) received intramuscular injection of 1 x 1011 vg (vector genomes) of
AAV1-hFIX or 6x1010 vg of AAV2-hFIX vectors as described. Twenty-four hours after
AAV injection, cells from the draining lymph nodes and spleen of the mice were
harvested for detection of cell surface markers by flow cytometry. The size of draining
lymph nodes and spleen in the AAV1-injected and AAV2-injected mice were similar to
those in the naïve, untreated mice. We also observed no change in the number of
CD11c+ cells in the draining lymph nodes and spleen of AAV1 or AAV2-injected mice
when compared to naïve, untreated mice (Figure 2A). Baseline levels of cells expressing
CD11c remained around 2% in draining lymph nodes and around 5-6% in the spleen in
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both experimental and untreated groups. We observed no statistically significant
difference in the expression of co-stimulatory molecules on the CD11c+ cells in both the
draining lymph nodes and spleen of AAV1 and AAV2-injected mice when compared
with naïve untreated mice (Figure 2B). In summary, we did not observe significant upregulation in the numbers or activation of DCs after intramuscular injection of AAV.
LPS and Anti-CD40 antibody can efficiently recruit DCs and induce complete
activation of DCs
To further validate our preliminary observation and hypothesis, we proposed to
investigate the effect of DC activation on immune responses to FIX following
intramuscular injection of AAV1. Intramuscular injection of AAV alone does not affect
the number and activation status of DCs. Therefore, we injected mice with LPS and antiCD40 antibody to activate DCs as described. Anti-CD40 antibody has been proven to be
a potent activator of DCs, B cells, epithelial cells, etc, causing strong inflammation.22,23
Cohorts of eight to ten week old C57BL/6 mice received intramuscular injection of 1 x
1011 vg of AAV1-hFIX and 6 x 1010 vg of AAV2-hFIX with or without administration of
LPS and anti-CD40 antibody (n=4 for each cohort). Twenty-four hours after
intramuscular injection of AAV1/AAV2 and/or administration of LPS and anti-CD40
antibody, cells from the draining lymph nodes and spleen of the mice were harvested for
detection of cell surface markers by flow cytometry. The size of draining lymph nodes
and spleen in the mice that received LPS and anti-CD40 antibody were considerably
larger when compared to naïve, untreated mice, or the mice that received only
intramuscular injection of AAV. We observed a significant increase in the number of
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CD11c+ cells in both the draining lymph nodes as well as spleen of mice that received
LPS and anti-CD40 antibody when compared to naïve and untreated mice (Figure 3A).
Significant expression of MHCII and co-stimulatory molecules (CD80 and CD86) was
also detected in the CD11c+ cells in both the draining lymph nodes and spleen of the
mice that received LPS and anti-CD40 antibody, compared to naïve and untreated mice
(Figure 3B). These results demonstrated significantly increased numbers and complete
phenotypic activation of DCs in experimental mice following a single administration of
LPS and anti-CD40 antibody.
Upon activation, DCs express pro-inflammationary cytokines. We thereby
examined cytokine expression of the activated DCs to further validate the complete
activation of DCs following administration of LPS and anti-CD40 antibody .We analyzed
the expression of cytokines in these cells by flow cytometry. We observed that there was
a significant increase in the number of CD11c+ cells expressing proinflammatory
cytokines IL-6 and IL-12 after administration of either LPS and anti-CD40 antibody
(p=0.0111 for IL-12, p= 0.0139 for IL-6) or anti-CD40 antibody alone (p= 0.0030 for IL12) in comparison to CD11c+ cells from naïve and untreated mice (Figure 3C). However,
there was no difference in the expression of IL-10 amongst the experimental and naïve
groups. This further validated complete activation of these DCs by LPS and anti-CD40
antibody.
Activation of DCs does not abrogate the FIX tolerance induced by intramuscular
injection of AAV1
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In steady state without inflammatory mediators, DCs are known to capture,
process and present antigen to T cells leading to immune tolerance. Upon inflammatory
stimulation, DCs are activated to initiate and promote immunity. We thereby deduced
that induction of inflammation and DC activation at the same time as AAV injection
would have the potential to change the immunological outcome following intramuscular
injection of AAV. We then evaluated the effect of DC activation on FIX tolerance
induced by intramuscular injection of AAV1 vector. Following intramuscular injection
of AAV1 with or without co-administration of LPS and anti-CD40 antibody, we
examined levels of human FIX antigen and anti-hFIX antibody in the circulation of the
experimental mice . Low levels of anti-hFIX antibody, and equivalent levels of
circulating hFIX antigen were detected in the mice regardless of the activation of DCs by
co-administration of LPS and anti-CD40 antibody (Figure 4A-B, n=5 for each cohort).
The levels of anti-hFIX antibody in mice that received LPS and anti-CD40 antibody are
comparable to those that did not receive LPS and anti-CD40 antibody. We are aware of
the detection of low levels of anti-hFIX antibody in mice after intramuscular injection of
AAV1, which are slightly higher than background anti-hFIX antibody in naïve isogenic
mice. Such low levels of anti-hFIX antibodies do not exhibit inhibitory activity.15 We
also observed that activation of DCs does not exaggerate the immune response induced
by intramuscular injection of AAV2. Eight to ten week old C57BL/6 mice received
intramuscular injection of 6 x 1010 vg of AAV2-hFIX with or without anti-CD40
antibody (n=5 for each cohort). We observed barely detectable levels of hFIX antigen
levels in both experimental groups. Very high levels of anti-hFIX antibodies were
observed which was further increased on immunogenic challenge (Figure 4C-D).
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Furthermore, no change of hFIX antigen levels and anti-FIX antibodies titers in
the AAV1 and DC activator-injected mice after immunogenic challenge of rhFIX
emulsified in CFA, demonstrated definite immune tolerance to FIX in these mice (Figure
4A-B). On the other hand, injection of rhFIX/CFA in AAV2-injected mice, irrespective
of whether they received anti-CD40 antibody or not, increased the titers of anti-FIX
antibodies (Figure 4C-D). These results clearly demonstrate that activation of DCs cannot
affect the immunological outcome of FIX tolerance induced by intramuscular injection of
AAV1.
We also examined expression of FIX antigen at early time points after
intramuscular injection of AAV1-FIX. We detected human FIX antigen in AAV1injected mice as early as 24 hours after intramuscular injection of AAV1-FIX, which
became significant by day 5 (Mean=165.3+/-SEM=55.27 ng/ml, n=5). Detection of
immediate expression of FIX antigen at early time points post AAV injection
demonstrates exposure of the host immune system to the FIX antigen when DCs are
activated by the inflammatory stimulation of LPS and anti-CD40 antibody.
DC activation-independent FIX tolerance induced by intramuscular injection of
AAV1 is irrespective of genetic and MHC backgrounds of mice
It was reported that mice with C57BL/6 are more tolerogenic than other strains of
mice such as Balb/c and C3H.24,25 We then moved on to test whether activation of DCs
could reverse the immune outcome in mice with different genetic and MHC backgrounds
by using Balb/c (H2d) and C3H (H2k) mice. Eight to ten week old Balb/c and C3H mice
received intramuscular injection of 3 x 1011 vg of AAV1-hFIX (a dose required to induce
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FIX tolerance)13 alone or with LPS and anti-CD40 antibody (n=5 for each cohort). We
examined levels of human FIX antigen and anti-hFIX antibodies in the circulation of the
experimental mice. A peak in circulating FIX antigen levels were observed at 8 weeks
followed by a stable expression of FIX until week 16 in both Balb/c (Figure 5A) and C3H
mice (data not shown). A slight peak in anti-FIX antibodies was observed initially at
week 4 followed by very low levels until week 16 in both Balb/c (Figure 5B) and C3H
mice (data not shown). The levels of both circulating hFIX antigen and anti-hFIX
antibodies were comparable irrespective of whether the DCs were activated with LPS and
anti-CD40 antibody or not. DC activation failed to reverse the tolerance observed after
intramuscular injection of AAV1 (Figure 5 depict Balb/c results, C3H mice data not
shown). This indicates that activation of DCs does not change the immunological
outcome in mice with diverse genetic and immunological backgrounds.
Discussion
Adverse immune responses following gene transfer are considered one of the
major causes of the minimal success in gene therapy clinical trials to date.2-4 It is of great
importance to elucidate the effect and the extent of gene transfer vector on adverse
immunity for successful gene therapy. The nature of the gene transfer vectors is
suggested as a foundation accounting for undesirable immune responses subsequent to
gene transfer.1,2 Vectors, the majority of which are derived from viruses, are encountered
and recognized as “danger signals” by the host immune system. Such viral vectors cause
a certain extent of inflammation by stimulating and initiating activation of APCs, leading
to unwanted anti-vector and/or transgene product immunity.1,2 Very little is known about
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the exact nature of APCs that are implicated in the initiation of anti-hFIX immune
responses subsequent to intramuscular injection of AAV.
DCs are the most potent and the major type of professional APCs with a unique T
cell stimulatory aptitude.6,7 DCs are the essential link between the innate and the adaptive
immune responses.7 The activation status of DCs has been proven to play a major role in
determining the immunological outcome. Immature or mature DCs in a steady state have
been shown to lead to T cell tolerance.6,9,10 The activation of the DCs and their function
in immune regulation, in turn, is dictated by the innate immune responses to different
pathogens and the consequent “danger signals”.6,7 Such innate immune responses and the
relevant “danger signal” triggers migration, maturation, differentiation and activation of
the DCs by significantly up-regulating expression of MHC and co-stimulatory molecules,
which are critical in initiating/promoting T cell priming and proliferation for effective
immunity.6 Activation of DCs not only precedes adaptive immune responses, it also
determines and controls the final outcomes of the immune responses.6
The non-pathogenic AAV vectors elicit negligible inflammation and thus have
minimal effect on DC activation.11,12 It is conceivable that inefficiency in activation of
DCs would favor lower immunity against vector as well as the transgene product. It was
reported that there was insignificant immunity subsequent to AAV gene transfer.26 We
hypothesized that inefficiency of DC activation subsequent to AAV gene transfer is a
factor that promotes FIX tolerance.
The objective of this study was to investigate the effect of activation status of the
major APC on humoral immune responses to FIX (formation of inhibitory anti-FIX
antibodies or FIX tolerance) following intramuscular AAV gene transfer for hemophilia
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B treatment. The results of our studies first revealed absence of, or inefficient
recruitment and activation of DC subsequent to intramuscular AAV gene transfer. This is
consistent with the non-pathogenic nature of the AAV vector, thus in support of our
hypothesis.
If the absence of DC activation were essential for induction of FIX tolerance in
the context of intramuscular injection of AAV, activation of the DCs would abrogate the
FIX tolerance and elicit anti-FIX immunity. We however observed efficient induction of
immune tolerance to FIX subsequent to intramuscular injection of AAV1 regardless of
full phenotypic and functional activation of DCs. Such an observation is contrary to our
hypothesis and is also inconsistent with the danger signal theory.
Accordingly, complete activation of dendritic cells locally and systemically,
failed to initiate an anti-FIX immunity or obliterate the immune tolerance to FIX induced
by intramuscular AAV1 gene transfer. This indicates the likelihood of the limited role
the effect of inflammation, innate immunity and the subsequent activation of DCs plays
in determining the immune responses to transgene product in the context of intramuscular
gene transfer. It also suggests that the non-pathogenic nature of AAV vectors and the
consequent minimal inflammation of AAV gene transfer may not contribute as much to
induction of immune tolerance to the transgene product, as postulated. In support of our
results, immune tolerance to FIX was also observed in FIX gene transfer using adenoviral
and lentiviral vectors targeting the liver.16,27 However, significant inflammation and DC
activation was observed subsequent to gene transfer of adenoviral and lentiviral
vectors.2,16,27 This also seemed to be inconsistent with the “danger signal” theory of
immunology.2,5,6 A body of recent experimental evidence strongly suggests that
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maturation of DCs can also render them to be tolerogenic.6,9,10 We would like to point
out that the LPS and anti-CD40 antibody-stimulated DCs in this study, are not only fully
matured, but also completely activated. Administration of anti-CD40 antibody also
causes a substantial level of general inflammation in addition to complete activation of
many types of APCs such as DCs, B cells, even endothelial cells.22,23 To our knowledge,
there is no report or proposal suggesting tolerance induction under the condition of
complete activation of DCs.
Unique characteristics of gene transfer may make immune responses to trangene
product in the context of gene transfer distinctive from the immunology of classical
protein replacement. As for the immune responses to FIX following intramuscular
injection of AAV vector, multiple factors including property of the gene transfer vectors,
vector dose, targeting organ/tissue, efficiency and amount of the transgene product
expressed, persistent expression of the transgene product, etc. may play a certain part in
determining the ultimate immunological outcome. High levels of FIX antigen were
reported as critical in deciding the immune responses to FIX following muscular AAV
gene transfer.13-15,18 It is however elusive as to whether there is a single decisive factor, or
a synergy of several factors that command the eventual immunological outcome in the
context of AAV gene transfer. Our results in the current study clearly demonstrated that
activation of DCs alone could not reverse induction of FIX tolerance in the context of
intramuscular injection of AAV vectors. Ongoing efforts in our laboratory are
investigating the interaction among multiple factors such AAV dose, antigen (FIX) level,
DC differentiation status, etc in deciding the ultimate immunological outcome to FIX in
the context of intramuscular AAV gene transfer.
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It was also reported that the inefficiency of local inflammatory activation by AAV
at injection sites could be overcome by an increase of “adjuvant”-like component(s) in
the AAV vector preparation upon elevated dose of AAV vectors.20 It was proposed that
the intensified local inflammation at the AAV injection site consequently led to higher
risk of formation of anti-FIX antibodies.20 Inefficient expression of adequate levels of
FIX antigen rather than the local inflammation caused by high dose AAV seemed to play
a major role in the higher risk of anti-FIX antibodies formation in that report.13,15,18
In summary, we demonstrated that activation of dendritic cells could not abrogate
the FIX immune tolerance induced by intramuscular injection of AAV1 vectors. Our
results provided critical insight into not only the immune responses to the transgene
product and the relevant mechanisms following gene transfer, but also the universal
principles and mechanisms governing general immune responses. Better understanding
of the mechanisms involved in the immune responses to FIX following gene transfer will
facilitate development of a successful gene therapy approach for hemophilia treatment
and FIX tolerance induction.
Acknowledgements: The authors claim no conflict of interest. This work is supported
by grant NIH R01-HL076699 to HJC. HJC is an NHLBI/NHF researcher.
Author contributions: HJC designed the study, analyzed the data and wrote the paper.
AB, MK and DK performed the experiments. AB summarized the data and participated
in data analysis and paper writing.
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Kelly ME, Zhuo J, Bharadwaj AS, Chao H. Induction of immune tolerance to FIX
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Figure Legends
Figure 1. Kinetics of dendritic cell activation upon administration of LPS and antiCD40 antibody. Eight to ten week old C57BL/6 mice (n=4 per cohorts) received LPS
(100μg/mouse) plus anti-CD40 antibody (100μg/mouse) (red line) or anti-CD40 antibody
alone (blue line) or phosphate buffered saline (PBS) (green line). Cells from draining
lymph nodes (A) and splenocytes (B) were collected at 6, 24, 72, and 120 hours after
injection and stained for cell surface antigens of CD11c (top panel), CD80 (middle panel)
and CD86 (bottom panel). The cells were first gated on the CD11c+ population of live
cells. Expression of CD80 and CD86 were determined from the gated CD11c population.
A representative of 2 independent experiments for each time point is shown.
Figure 2. Direct intramuscular injection of AAV vectors does not activate DCs. Eight
to ten week old C57BL/6 mice (n=4 per cohorts) received intramuscular injection of
1x1011 vg of AAV1-hFIX or 6x1010 vg of AAV2-hFIX. Control mice of similar age were
administered phosphate buffered saline (PBS). After 24 hours, cells from the draining
lymph nodes and spleen were collected and stained for the appropriate antibodies and
flow cytometry analyses were performed as described.
(A) No change in DC numbers in draining lymph nodes and spleen following
intramuscular injection of AAV. The dot plots show the expression of CD11c on
cells. Numbers indicate the percentage of CD11c+ cells amongst the total live
cells in either the draining lymph nodes or spleen.
(B) No activation of DCs in draining lymph nodes and spleen following
intramuscular injection of AAV. The dot plots show the expression of either
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MHCII and CD80 or MHCII and CD86 on cells gated with CD11c. Numbers
indicate the percentage of MHCII+CD80+ or MHCII+CD86+ amongst the
CD11c+ population. Data shown are mean ± SEM. A representative of 2
independent experiments is shown.
Figure 3. Anti-CD40 antibody and LPS recruits and activates DCs in the draining
lymph nodes and spleen. Eight to ten week old C57BL/6 mice (n=4 per cohorts)
received intramuscular injection of 1x1011 vg of AAV1-hFIX + 100μg of LPS + 100μg of
anti-CD40 antibody (in the footpad) or 6x1010 vg of AAV2-hFIX + 100μg of LPS +
100μg of anti-CD40 antibody (in the footpad) on the same day. Control mice received
phosphate buffered saline (PBS). Cells from the draining lymph nodes and spleen were
collected after 24 hours, stained with the appropriate antibodies and flow cytometry
analysis was performed as described.
(A) Increase in DC numbers in draining lymph nodes and spleen following
administration of LPS and anti-CD40 antibody in comparison to naïve mice.
The dot plots show the expression of CD11c on cells. Numbers indicate the
percentage of CD11c+ cells amongst the total live cells in either the draining
lymph nodes or the spleen.
(B) Activation of DCs in draining lymph nodes and spleen following
administration of LPS and anti-CD40 antibody. The dot plots show the
expression of either MHCII and CD80 or MHCII and CD86 on cells gated
with CD11c. Numbers indicate the percentage of MHCII+CD80+ or
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MHCII+CD86+ amongst the CD11c+ population. Data shown are mean ±
SEM. A representative of 2 independent experiments is shown.
(C) Cytokine profile of splenocytes after 24 hours following administration of
LPS and anti-CD40 antibody. The histograms show the expression of IL-12
(top panel), IL-6 (middle panel) and IL-10 (bottom panel) on cells gated with
CD11c. Numbers indicate the percentage of CD11c+ cells that are expressing
the particular cytokine. Data shown are mean ± SEM. n= 4 for each cohort. A
representative of two independent experiments is shown.
Figure 4. Activation of Dendritic cells does not abrogate FIX tolerance induced by
intramuscular injection of AAV1 in C57BL/6 mice. Plasma was collected every 4
weeks after AAV and LPS + anti-CD40 antibody injection, and measured for hFIX
antigen and anti-hFIX IgG antibodies by ELISA. Data shown are mean ± SEM. n=5 for
each cohort.
(A) Human FIX antigen. Eight to ten week old C57BL/6 mice received
intramuscular injection of 1x1011 vg of AAV1-hFIX alone (Closed square) or
with LPS and anti-CD40 antibody (Open square).
(B) Anti-hFIX IgG antibodies. Eight to ten week old C57BL/6 mice received
intramuscular injection of 1x1011 vg of AAV1-hFIX alone (Closed square) or
with LPS and anti-CD40 antibody (Open square).
(C) Human FIX antigen. Eight to ten week old C57BL/6 mice received
intramuscular injection of 6x1010 vg of AAV2-hFIX alone (Closed square) or
with anti-CD40 antibody (Open square).
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(D) Anti-hFIX IgG antibodies. Eight to ten week old C57BL/6 mice received
intramuscular injection of 6x1010 vg of AAV2-hFIX alone (Closed square) or
with anti-CD40 antibody (Open square).
Figure 5. Activation of DCs does not abrogate FIX tolerance induced by
intramuscular injection of AAV1 in Balb/c mice. Eight to ten week old Balb/c mice
received intramuscular injection of 3x1011 vg of AAV1-hFIX alone (Closed square) or
with LPS and anti-CD40 antibody (Open square) (n=5 for each cohort). Plasma was
collected every 4 weeks after AAV and LPS + anti-CD40 antibody injection, and
measured for hFIX antigen (A) and anti-hFIX IgG antibodies (B) by ELISA. Data shown
are mean ± SEM.
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Prepublished online November 17, 2009;
doi:10.1182/blood-2009-08-239509
Induction of immune tolerance to FIX by intramuscular AAV gene transfer
is independent of the activation status of dendritic cells
Arpita S. Bharadwaj, Meagan Kelly, Dongsoo Kim and Hengjun Chao
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