Blood, 15 August 2002, Vol. 100, No. 4, pp. 1133-1140
REVIEW ARTICLE
Dangerous liaisons: the role of "danger" signals in
the immune response to gene therapy
Brian D. Brown and
David Lillicrap
From the Department of Pathology, Queen's
University, Kingston, Ontario, Canada.
 |
Abstract |
Recent studies in gene transfer suggest that the innate immune
system plays a significant role in impeding gene therapy. In this
review, we examine factors that might influence the recruitment and
activation of the innate system in the context of gene therapy. We have
adopted a novel model of immunology that contends that the immune
system distinguishes not between self and nonself, but between what is
dangerous and what is not dangerous. In taking this perspective, we
provide an alternative and complementary insight into some of the
failures and successes of current gene therapy protocols.
(Blood. 2002;100:1133-1140)
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Introduction |
According to the immunological theory of
self-nonself (SNS), peptides that are not present during early ontogeny
can be expected to be treated as foreign by the immune system. This
axiom would predict the rejection of transgene products introduced by
gene therapy for monogenic disorders in which individuals are deficient for a particular protein. Given the minimal success of gene therapy to
date, owing in part to host immune responsiveness, this hypothesis would appear to be supported. Many of the studies thus far have shown a
common pattern in which the immune system initially attacks the
delivery vector and subsequently responds to the transgene product.1 Assays for antibodies against the new protein,
as well as the measurement of cytotoxic T-lymphocyte (CTL)
responsiveness, have been demonstrated, and have been used to explain
the lack of success in these trials.2,3
There may, however, be an alternate hypothesis to explain these
observations. Rejection may not be due solely to the "foreignness" of the transgene, but instead may be due, at least in part, to the
"danger" associated with the gene delivery process and the synthesis of the new transgene product. A novel theory of immunology proposed by Matzinger4 suggests that the immune system
does not distinguish between self and nonself, but between dangerous and not dangerous. This notion may have important consequences in the
field of gene therapy, where host immune responses may be one of the
most significant barriers to success. There are several key danger
signals encountered in gene therapy, including the vector, DNA, local
inflammation, and endogenous cellular signals. We propose that these
signals initiate the immune response against the transgene, as well as
the transgene product, and result in the failure of many gene therapy
protocols. In this review we discuss the relevance of the danger theory
as it pertains to the immunologic response observed in gene therapy. In
the interest of space, we focus our attention on therapies targeting
monogenic disorders. For a good review that relates the danger model to gene-based strategies for cancer therapy, refer to Van Tendeloo et
al.5
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APC maturation and T-cell activation |
Dendritic cells (DCs) are a type of antigen-presenting cell (APC)
that can be found in most tissues throughout the body.6 They reside in an immature state in which they have high concentrations of Fc
and Fc
receptors on their cell surface, and have been shown, in vitro, to be actively involved in phagocytosis and
macropinocytosis, a process that enables sampling of the extracellular
environment for solutes.7-10 In this state they present
only very low levels of major histocompatibility (MHC) molecules and
other cell surface markers such as CD40, CD54 (intercellular adhesion
molecule-1 [ICAM-1]), CD58 (lymphocyte function-associated antigen-3
[LFA-3]), CD80 (B7.1), and CD86 (B7.2).11,12 Normally
quiescent, they begin to migrate through a tissue in response to a
barrage of cytokines that include tumor necrosis factor (TNF)-
,
interleukin (IL)-1
, interferon (IFN)-, macrophage inflammatory
protein (MIP)-1, and MIP-1.9,13-15
Once they enter a site, DCs and other APCs, such as macrophages, can
take up particles by phagocytosis and, in so doing, begin to
mature.12 This process diminishes the APC's ability to
further endocytose molecules and allows the cells to begin presenting peptides on MHC class I and MHC class II molecules.12 The
maturation period occurs over approximately 24 hours, and during this
time the APC begins to move to lymphoid organs such as the spleen and draining lymph nodes. Inside lymphoid organs, APCs are exposed to
millions of naive T cells.16 The MHC-peptide complex
presented by the APC is allowed to contact individual T-cell receptors
(TCRs) in an effort to locate a TCR capable of recognizing the peptide antigen being presented. The interaction of MHC-antigen with an appropriate TCR is the first step in initiation of an immune response and is referred to as signal 1.17
Signal 1 alone, however, is not sufficient to activate a naive T
cell.18 A second set of signals, which will collectively be designated as signal 2, must also occur to initiate the T-cell response. Signal 2 involves the interaction of adhesion and
costimulatory moleculeson the APC cell surface with the T
cell.19,20 In Table 1, we
list some of the molecules involved in signal 2. This list is not
comprehensive, and it is important to note that the details of how
these molecular interactions affect the T cell are still not well
understood. What we do know is that signal 1 in the presence of signal
2 will activate a naive T-cell clone capable of recognizing the
presented epitope, and that the result will be an immune response directed against the source antigen (Figure
1).4 In contrast, if a naive
T cell receives signal 1 without signal 2, the T cell will be
down-regulated (anergy) or deleted (apoptosis).4 For a
productive signal 2 to be generated, APCs must first be activated. This
has important consequences for gene therapy because it predicts that an
immune response against the transgene or transgene product will occur
only if APC activation has occurred. The mere presence of any antigen,
including a "neo-antigen" created by a transgene, is not sufficient
in itself to provoke a response. Costimulation is necessary, and the
question therefore is, what is the signal (signal 0) that induces the
up-regulation of signal 2 on APCs?
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| Figure 1.
APC antigen presentation to naive CD4+ cell.
(1) APCs presenting antigens on MHC class II molecules (signal 1) are
stimulated to express costimulatory molecules (signal 2) by endogenous
and exogenous factors (signal 0)  naive T cells receiving signal 1 in the presence of signal 2 are activated and an immune response is
initiated against the antigen. (2) An APC presenting antigen (signal 1)
and stimulated by signal 0 to express costimulation (signal 2) does not
find a T-cell receptor (and thus a naive T cell) capable of recognizing
the presented antigen T cell previously deleted or down-regulated
(anergy) when encountering the same (or similar) antigen without signal
2. (3) A naive T cell receiving signal 1 in the absence of signal 2, by
an APC, is deleted or anergy is induced.
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Danger signals |
We believe that there are certain danger signals inherent to gene
therapy that are capable of acting as signal 0 and activating the APCs
(Figure 2). The most significant of these
danger signals encountered to date has been the delivery vehicle. The
majority of the delivery systems currently being used are viral-based
vectors that have been constructed by modifying pre-existing virus
genomes and that require packaging in viral capsids. Unfortunately for gene therapy, the host tissues have, over time, learned to recognize many of these viruses and treat them as dangerous. This recognition occurs without any input from the adaptive branch of the immune system
and is inherent to most tissues of the body.
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| Figure 2.
Danger-signal molecules capable of activating
antigen-presenting cells.
(A) Endogenous danger signals are molecules originating from the host
organism; these are products generally released during events of
cellular stress. Two general categories of endogenous signals exist:
(1) molecules that are secreted by stressed cells such as cytokines,
and (2) intracellular products released when membrane disruption occurs
(necrosis). For a comprehensive review of these signals and their
corresponding receptors, refer to Gallucci and
Matzinger.27 (B) Exogenous danger signals include a vast
array of molecules associated with pathogenic organisms. For a
comprehensive review of these signals and their corresponding
receptors, refer to Aderem and Ulevitch.28
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During a pathogenic infection, a tissue becomes stressed and begins to
secrete soluble factors such as granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1, TNF-, and IFN-, which cause local inflammation and recruit cells of the innate immune system,
including DCs and macrophages.29-32 Evidence of this
phenomenon occurring during gene therapy has been demonstrated in a
number of experiments in which levels of these cytokines were shown to rise within hours of vector administration.33-36 The
initiation of this process can best be understood in the context of the
danger model of immune responsiveness. Over millions of years of
evolution, and before the development of the adaptive response, the
immune system evolved a primitive method of recognizing pathogenic
invaders. Recognition does not occur through the vast repertoire of
TCRs or antibodies, but instead by pattern-recognition receptors, which identify common structures on pathogens.28,37
Although many pattern-recognition receptors have yet to be identified,
there have already been some described that recognize lipopolysaccharide from Gram-negative bacteria and peptidoglycans on
Gram-positive bacteria and yeast.38-42 There is even a
class of these molecules known as toll-like receptor 4 (TLR4), which has been shown to be activated by viral proteins.43 These
receptors are clearly a mechanism evolved not to recognize nonself, as
they are too limited in their diversity, but to identify molecules commonly associated with dangerous or harmful organisms.
In addition to viral and bacterial proteins, other substances can
trigger the toll-like receptors and act as the initiators of signal 0. One of the most significant of these signals, in relation to gene
therapy, is the transgene DNA itself.44 Mammalian DNA
differs from its prokaryotic counterpart in its degree of CpG
methylation.45 The innate system has learned to recognize these differences and can be activated in the presence of unmethylated CpG sequences.46 Experiments have been carried out to
compare plasmid vectors with reduced numbers of CpG
dinucleotides.47 The removal of these sequences was
accomplished either by elimination of nonessential sequences or through
site-directed mutagenesis. When the plasmids were injected into mice,
the animals receiving vectors with a reduced number of CpG motifs
experienced a reduction of up to 75% in their serum levels of the
inflammatory cytokines IL-12, IFN-, and IL-6. This suggests an
important variable to be considered when trying to either abrogate or
stimulate an immune response.
There is also an important class of danger signals that are not
directly related to the pathogen. These are normally found only within
the intracellular environment and are released exclusively following
necrotic, as opposed to apoptotic, cell death. Gallucci et
al48 have performed experiments in which they administered either necrotic or apoptotic cells to DCs in culture and in mice, as
adjuvants. The DCs were then evaluated for cell surface expression of
the costimulatory molecules B7.1 and B7.2, as well as MHC class I and
class II. Gallucci et al found that only the necrotic cells were
capable of activating the DCs. It therefore appears that the body has
devised a method of initiating APC activation under conditions of
stress, such as when cells die unexpectedly. This observation further
emphasizes the principle that the innate immune system has learned to
respond only when harmful circumstances are present.
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A "dangerous" therapy |
In many gene therapy regimens, danger signals are being introduced
in conjunction with the introduction of the new transgene. A viral
vector invades the host cells and inflammatory signals are generated,
resulting in the recruitment of the innate immune system and subsequent
cell death. The local DCs scavenge for antigens, including those that
are being produced by the transgene, and they become activated. The DCs
are now capable of presenting antigen with signal 2 to naive T cells
and initiating an immune response against the therapeutic transgene and
transgene product.
Of course, the mature DCs will also carry antigens belonging to the
host, but T cells able to recognize a host MHC-antigen complex would
have been deleted or "anergized" in either the thymus or the
periphery when they previously encountered the antigen on an APC that
was not activated. Therefore, only T-cell clones specific for the
transgene and the delivery vehicle, which have not been seen
previously, will be recruited to the site of vector delivery.
Consequently, an immune response directed against the therapeutic
protein and the cells producing the protein and any viral genes will occur.
From this description and the mechanisms represented in Figure 1, it
can be concluded that the danger theory and the theory of self-nonself
are not mutually exclusive, but rather alternative and complementary
principles for explaining specific immunologic responses. Signal 1, the
antigen, can still be regarded as self or nonself, but the danger
theory suggests that an additional regulatory mechanism exists by
asserting that the context in which the antigen is presented, danger or
steady state, is a critical factor that determines how the immune
system will respond to the antigen. Thus, unlike the SNS model, which
predicts that the body should never accept a new gene and its protein
product, the danger model predicts that immunologic tolerance can occur
if the danger is removed from the gene delivery process.
Support for this model is provided by experiments in which similar
genes are introduced with adenovirus and adeno-associated virus (AAV)
and 2 different immune responses are observed.49 In
animals that receive the adenovirus vector, a CTL response ensues
against those cells expressing the transgene (an observation confirmed
by our laboratory
see below). In contrast, no CTL response is observed
in animals that receive the transgene by AAV delivery. According to the
SNS model, it is the "foreignness" of the gene product that is
immunostimulatory, regardless of the delivery vehicle, and introduction
of the gene product should therefore result in an immune response with
either vector. The danger theory, however, makes no such prediction,
and as we discuss below, it even provides an answer for this biological conundrum.
Jooss et al50 have shown that AAV does not transduce APCs
as efficiently as adenovirus and therefore minimizes signal 1. In
addition, AAV may also minimize the occurrence of signal 0. AAV is a
nonpathogenic virus and does not, in itself, represent a potential
danger to the target organism.51 The innate system may not
have evolved pattern recognition receptors to recognize AAV capsid
proteins, which may explain the minimal level of inflammation observed
during AAV infection. In addition, expression of transgenes delivered
by AAV may be delayed by as long as several weeks after initial
infection.52-54 This allows the immune system time to
clear away antigens and adjuvants in the localized area of vector
delivery before the therapeutic protein reaches the extracellular
environment. The new protein is thus presented in a nondangerous
setting in which APCs are not activated. T cells would therefore
receive signal 1 without signal 2, and a state of tolerance for the new transgene protein would occur.
In contrast, protocols utilizing adenovirus as the delivery vehicle
induce a much different response in the host. Within hours of
infection, levels of inflammatory cytokines, including IL-6 and
TNF-, begin to increase.33-36,55 This is closely
followed by activation of DCs and macrophages, as shown by increased
measurement of the cell-surface costimulatory molecule, CD86, on these
cells.56 Furthermore, at high vector doses, alanine
transaminase serum concentrations are elevated more than 50-fold over
those of control animals within the first 24 hours, indicating
significant hepatotoxicity.57 The consequence of this
"stressed" environment is the activation of innate immunity and the
subsequent induction of humoral and cellular immunity directed against
the viral vector, the therapeutic protein, and even the host cells
harboring the delivered transgene.2,3,49,58
An interesting adjunct to this hypothesis is provided by AAV
experiments in which a humoral response is observed against the viral
capsid and even the transgene product.59,60 Although AAV
infection is not highly inflammatory, other danger signals, such as
IFN- expression by the target tissue or cell necrosis, may act as a
signal 0. This would serve to recruit and activate APCs capable of
scavenging antigens at the site of infection. Viral capsids would be
present, and it has been suggested that contaminating transgene product
may also be present at the time of gene delivery that was copurified
with the vector. Fortunately, necrotic cell debris and other danger
signals dissipate before host cells begin expressing the
transgene. Hence, only a transient humoral immune response would
be anticipated against the exogenous protein and viral capsid.
The immune response generated against stably integrating
vectors, such as oncoretrovirus and lentivirus, can also be
predicted in the context of the danger theory. Although many of these
viruses have not been shown to cause human disease or significant
toxicity, which may explain their partial success in mediating
long-term transgene expression, their use has still been limited, in
part, by host immune responses.1,61-65
Human immunodeficiency virus (HIV)-based lentiviral vectors are a
class of retroviral vectors capable of infecting and integrating into
dividing and nondividing tissues.66 Experiments were
performed in which portal vein injections of a lentiviral vector
containing a human factor VIII (hFVIII) cDNA were administered to
C57Bl/6 mice following a partial hepatectomy.67 FVIII
levels reached 30 ng/mL (~ 15% of normal), but the elevation was
transient, and the subsequent drop in FVIII levels was accompanied by
the appearance of anti-hFVIII inhibitors. While several different
variables may have influenced the development of immunity, the partial
hepatectomy would undoubtedly have involved the recruitment of the
innate immune system. This procedure, which was undertaken to optimize lentivirus transduction, results in high levels of cell necrosis that
can stimulate APC activation and may have been responsible, at least in
part, for the induction of inhibitor formation.27
It is interesting to note that in the same study, by Park et
al,67 when human factor IX (hFIX) was used as the
transgene, inhibitors did not develop against the therapeutic protein
even though these animals also underwent partial hepatectomy. This observation may appear contrary to the prediction of the danger theory;
however, as we have already indicated, when a protein is present in the
absence of danger signals, tolerization may be expected. hFIX shares a
significantly higher degree of sequence homology with murine
FIX68 than hFVIII shares with murine FVIII.69 As a result, prior deletion of T-cell clones capable of recognizing similar epitopes on the mouse and human molecules would occur during
periods without danger signals, and the animal would be left with a
limited ability to respond to the hFIX transgene protein. Thus, the
nature of the transgene product, even in the context of the danger
theory, is still a critical mediator of the immune response, as it
provides the source of signal 1.
The ability of retroviruses to integrate into target genomes may
provide a further advantage to these vectors. During initial infection
some level of danger may be anticipated from the administration procedure. This would be expected to stimulate APCs and, in turn, initiate a T-cell response against virus-infected cells. However, activated T-cell lifespan is finite, and these cells would soon undergo
preprogrammed cell death.70 Because the danger signals would have subsided, new naive T cells would not be activated and
recruited to the site of vector delivery, and memory T cells would be
anergized by unstimulated APCs presenting the therapeutic antigen.71 Hence, the stable integration of the transgene
would enable it to be propagated in tissues recapitulating themselves after immune system destruction, and therefore would allow for long-term expression of the transgene product.
This may also provide an explanation for the FVIII tolerance recently
observed by Chao and Walsh.72 Using an integrating AAV
vector to deliver hFVIII to mice, they demonstrated anti-hFVIII inhibitor formation occurring within 2 weeks of treatment. However, there was a subsequent rise in plasma FVIII levels 10 months after initial transgene delivery that correlated with the disappearance of
FVIII-specific antibodies. These results have significant implications for the treatment of disorders in which antibody formation is a common
complication of patient treatment.
There are also data implicating danger signals as one of the mechanisms
involved in the inactivation of viral promoter elements. In studies
comparing transgene expression mediated by the transcriptional regulatory elements from cytomegalovirus, Rous sarcoma virus, simian
virus 40, and the Muloney murine leukemia virus long terminal repeat
with the cellular -actin promoter, there is evidence to indicate
that TNF- and IFN- can mediate attenuation of the viral regulatory elements while having little effect on the endogenous cellular promoter.73 This is an important observation, as
it explains the tendency of viral promoters to be down-regulated in the
absence of a humoral or CTL response.74 The danger
model predicts that the tissues themselves are capable of recognizing potentially harmful agents or components of these agents, as would be
the case with a viral promoter. Thus an infected cell can trigger a
signal 0, such as INF-, to activate APCs to release TNF- and mediate the down-regulation of the viral promoter.
Methods of ex vivo gene therapy may provide a safer and less
immunogenic alternative to in vivo techniques by limiting the exposure
to danger signals. Viral peptides from delivery vectors introduced ex
vivo can be removed before the genetically modified cells are
reintroduced into the body. This results in reduced inflammation and
toxicity (signal 0) associated with the administration of many
pathogen-derived vectors and thereby limits mobilization and activation
of the immune system. Early experiments using terminally differentiated
cells including myoblasts,75,76
fibroblasts,77 and peripheral blood
lymphocytes78 indicated that immune-mediated rejection may
not be a significant barrier to successful implementation of ex vivo
strategies. However, retrospective analysis indicates many of these
protocols employed immunocompromised patients incapable of mounting an
immune response.79 Moreover, down-regulation of
therapeutic gene expression, due to transcriptional silencing, may have
limited the immunogenicity of the cellular grafts even before
introduction into the host.80
In experiments where genetically modified lymphocytes were selected for
sustained expression prior to reintroduction into patients,
T-cell-mediated immunity was in fact observed against the
grafts.79 This is not surprising, given that
differentiated cells do not induce tolerance but are capable of
presenting antigen in an MHC class I-restricted manner. Genetically
modified cells expressing a transgenic antigen could engraft in
immune-privileged sites or in the absence of immune system activation.
If, however, the genetically modified cells undergo necrosis, APCs
would be recruited by danger signals, resulting in APC maturation and
subsequent T-cell recruitment directed against the cells, as observed
by Riddell and colleagues.79
A more feasible method of ex vivo gene delivery may be provided by the
use of stem cells (SCs). Advances in isolation and culture conditions,
as well as improvements in transduction efficiency of stably
integrating vectors such as lentivirus, which are capable of infecting
nonproliferating cells, have increased interest in SC gene
therapy.81,82 These cells have been shown to offer significant advantages over the use of differentiated cell types and
may be capable of inducing specific tolerance to transgenic proteins.83-85 In experiments carried out by the Dunbar
laboratory, a retroviral neoexpression vector was delivered, ex vivo,
to both lymphocytes and hematopoietic stem cells (HSCs) and
subsequently reinjected into Rhesus monkeys.86 The
modified lymphocytes were quickly rejected by the host, whereas
transfer of the genetically modified HSCs resulted in long-term
engraftment and tolerance to the neopeptide. When further experiments
were carried out in which the tolerized animals were rechallenged with
lymphocytes carrying the neocassette, the cells were not rejected.
These results indicate that the HSCs were able to mediate persistent
tolerance even when the transgene was reintroduced in the context of an immunogenic delivery protocol.
Initial inoculation of genetically modified cells disrupts the body's
steady state and signals the innate immune system to respond. This may
explain why a humoral response directed against the components of fetal
calf serum is often observed immediately after infusion of modified
SCs.86,87 Over time, danger signals subside and the innate
system is no longer activated. SCs, unlike committed cells, begin to
differentiate into various tissues, including cells involved in antigen
presentation, such as DCs and macrophages.88 Under
steady-state, nondangerous, conditions, the transgenic antigen would be
presented to naive T cells in the absence of costimulation, inducing
T-cell anergy and resulting in antigen-specific tolerance.
This hypothesis predicts the success reported by Pawliuk et
al89 in which genetically modified HSCs were successfully
used to correct sickle cell disease for more than 10 months in 2 mouse models. While an erythroid-specific promoter was used to limit expression exclusively to this cell type, experiments with similar promoters have shown that expression can occur in 0.5% to 3.7% of
transduced B, T, and myeloid cells.90 We suggest the
possibility that antigen, presented by SC-derived transgenic APCs in
the absence of signal 2, induced tolerance to the A-T87Q-globin gene
variant and allowed for long-term engraftment and correction of the
disease phenotype.
Although SCs may offer significant advantages in the treatment of
monogenic disorders, their potential to induce a productive immune
response cannot be overlooked. SC-derived APCs under conditions of
stress would be capable of presenting the transgenic antigen, as well
as any donor-mismatched antigens produced by the therapeutic cells
(heterologous SCs), to naive T cells in the presence of costimulation.88 This would result in rejection of the
engrafted cells and/or humoral-mediated immunity against the transgene
product.87 The potential for these adverse events suggests
that investigators should minimize the likelihood of generating danger
signals when introducing SCs into a donor and should consider
monitoring stem cell differentiation and APC activation by
fluorescence-activated cell sorter (FACS) analysis of cell surface markers.
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Removing the "danger" |
Over the past 10 years, a significant effort has been under way to
reduce the immune response that arises during gene therapy protocols.
Unfortunately, very few of these attempts have been successful. We
believe the danger model offers insight into why some of these
techniques have failed and can provide predictions for the successful
manipulation of a host response.
Suppressing signal 1 is an obvious choice for blocking the immune
system's ability to respond to new genes and gene products. This
strategy has been employed in the field of transplantation medicine for
decades by using drugs such as cyclosporin and
tacrolimus.91 These agents inhibit the synthesis and
release of cytokines and prevent the differentiation of CD4 cells,
thereby blocking an immune response. Unfortunately, this therapy works
in a nonspecific manner and thus leaves the patient highly susceptible
to infections. In addition, as Matzinger71 has previously
noted, this is not a method of tolerization because it targets signal
1, and the patient must often remain on immunosuppressing drugs
indefinitely or risk rejecting the transgenic organ.
The danger theory predicts that tolerance to a molecule can occur when
a naive T cell is presented with signal 1 in the absence of signal
2.4 Therefore, blocking signal 2 during the period of time
when the transgene is first introduced could result in tolerance and
remove the need for permanent immunosuppression. To date, several
different strategies have been used to suppress signal 2. The most
commonly practiced techniques involve the use of CTLA4 immunoglobulin
(CTLA4Ig) and anti-CD40 ligand (-CD40L).92-95 These
molecules act to block important costimulatory pathways (Table 1) by
competing for and blocking T-cell receptors, thereby preventing naive
T-cell activation. In addition, CTLA4Ig can also turn off T-cell
production of IL-2, an important cytokine involved in the initiation of
cellular immunity.
More recently, a study by Jiang et al96 has shown
that CTLA4Ig and -CD40L, even when administered together, do not
induce a state of permanent tolerance. In this study, primary skeletal muscles were injected with a first-generation adenovirus vector containing an enhanced green fluorescent protein (AdEGFP) transgene in
conjunction with vectors expressing CTLA4Ig and -CD40L. This resulted in long-term expression and lack of anti-EGFP neutralizing antibodies. However, when a repeat injection of the AdEGFP vector was
given without the signal 2-suppressing vectors, a humoral response to
the EGFP developed in the animals.
We believe the failure of this approach occurs because readministration
of the vector reintroduces danger signals. There is no tolerance to
signal 0, and in the presence of the adenovirus and other inflammatory
stimuli the APCs will once again begin to mature. T cells previously
"anergized" by the initial treatment may now be activated by the
APCs. It is therefore necessary to coadminister signal 2 blockers for a
limited duration each time readministration is performed to block
T-cell activation and allow the danger signals to subside. The
importance of the viral DNA and protein capsid as adjuvants is
emphasized by work in which -CD40L was capable of inducing long-term
tolerance, even on repeat administration, when recombinant hFVIII
protein alone was given to hemophilic mice, and no humoral immunity
developed against the exogenous protein.97
The most critical element of the danger theory that differs from the
classic SNS model is its treatment of signal 0. Here the danger theory
predicts that by removing signal 0 the initiation of an immune response
can be avoided. Already there are many different techniques for
excluding potential danger signals. In the field of adenovirus-mediated
therapy, the most significant advance has been made by removing all the
wild-type genes from the virus.98-100 In our laboratory,
we have specifically compared the use of a 
E1E3-adenovirus with
the use of a helper-dependent vector (no viral coding sequences). In
these experiments, we have observed a CTL response directed against the
transgene delivered by the E1E3 vector, but no such response in
mice receiving the same transgene with the helper-dependent adenovirus
(B.D.B., F. Grant, unpublished observations, March 2002). The removal
of viral coding sequences has led to a reduction in immune
responsiveness and even long-term transgene expression, with some
"gutless" vectors demonstrating maintained therapeutic output for
up to 2 years after treatment.101-103
Similar gains have been made by using tissue-specific promoters
to drive transgene expression.104-106 Carrying out these
modifications has led not only to a decrease in viral and transgene
expression in APCs, but also to a reduction in danger signals supplied
with the delivery vehicle, including potentially immunostimulatory sequences present in viral promoters. Thus, the body's capacity to recognize those elements that are traditionally associated with
danger to the organism becomes limited, and the initiation of an immune
response is significantly less likely.
Some work has also been carried out using anti-inflammatory drugs
and cytokine modulating agents to reduce signal 0 in gene therapy.33,35,107,108 These reagents have shown benefit,
but they have not been entirely successful. One possible explanation for these failures may be that these protocols were used in
conjunction with early-generation vectors, often under the control of
viral elements. The use of these drugs with more current vector
systems, and under tissue-specific control, may help to increase their efficacy in mediating long-term transgene expression.
Recent evidence from Herzog et al109 suggests that
the immunosuppressive agent cyclophosphamide (CyP) may be capable of
inducing long-term tolerance. CyP does not specifically target signal 1 or signal 2, but instead acts in a systemic manner to interfere with
cell growth. Thus, it is capable of blocking the necessary cell
divisions required for a T- and B-cell response. In this report,
long-term correction of hemophilia B was observed in a dog receiving
intermittent, short-term treatment with CyP prior to, and following,
administration of an AAV vector delivering a canine FIX gene. There was
no humoral response observed against the FIX, but anti-AAV antibodies
did develop. Although the danger model does predict FIX tolerance in
this circumstance, as the use of CyP would serve to blunt the immune
response long enough for danger signals to subside and allow for APC
presentation of the FIX antigen in the absence of costimulation, the
development of anti-AAV immunity is unexpected. We are currently unable
to explain these observations in the context of the danger model (or
the SNS model), but we are aware that in addition to the vector components themselves, several other factors influence the development of immunity, including the route of administration, the target tissue/organ, the vector dose, the underlying genetic mutation, and the
species and strain of the recipient animal.1 Better comprehension of how these factors influence APC activation, and subsequent T-cell responsiveness, will help to provide insight into
these observations.
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Conclusion |
After a century of study, the fundamental mechanisms of immunity
and tolerance still remain elusive. In the 1940s and 1950s Burnet first
proposed the concept of self-nonself discrimination in his clonal
selection theory, which quickly became the central dogma of immunology
for much of the ensuing 40 years.110 However, over the
last decade an increasing number of questions have been raised about
this theory. This questioning has led to more current proposals of how
the immune system functions. Among the contemporary models of
immunology, which include an extension of the Jerneian idiotype network
theory by Coutinho, an expanded self-nonself theory by Medzhitov and
Janeway, an associative recognition model by Cohn and Langman, and the
antigen localization theory championed by Zinkernagel, we have chosen
to apply Matzinger's danger model to explain some of the observations
reported in gene therapy.111-114 This theory successfully
integrates current concepts in immunology with the growing literature
on molecular therapy.
We conclude this review with some words of caution. The immune system
involves a highly developed network of cells and regulatory elements
that must work together to protect the body without causing undue harm
to the individual. It is unlikely that any one theory is capable of
describing all observations related to this system. While we believe
that the danger theory offers a stronger model than the classic SNS
theory to describe some of the reported data concerning gene
replacement, as with all scientific theories, there is always room for
reevaluation as more experience is developed in this field of study.
We propose that, based on this model, future gene replacement
strategies should begin with the questions, are we administering something the body would have "historically" encountered as a threat? and will the gene delivery process result in a state of danger?
Answering these questions before proceeding will provide a better
prediction of the potential immunologic response to treatment and
hopefully enable the field of gene therapy to progress toward clinical
success in the most safe and efficient fashion possible.
| |
Acknowledgments |
The authors are grateful to Drs Peter Borgs, Christine Hough, Polly
Matzinger, and Thierry VandenDriessche for their critical review of
this article. We would also like to thank Ms Lisa Marie Picken for all
her support during the writing of this manuscript.
| |
Footnotes |
Submitted November 28, 2001; accepted March 29, 2002.
Prepublished
online as Blood First Edition Paper, May 17, 2002; DOI
10.1182/blood-2001-11-0067.
Supported by the Canadian Institutes of Health Research
(MT-10912), the Canadian Hemophilia Society, and the Bayer/Canadian Blood Services Partnership Fund.
Reprints: David Lillicrap, Department of Pathology, Queen's
University, Kingston, ON, Canada K7L 3N6; e-mail:
lillicrap{at}cliff.path.queensu.ca.
| |
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Abstract
Introduction
