<title>1. INTRODUCTION</title> <p>Organ
transplantation in the clinic became a reality in 1954 when Merrill, Murray,
and Harrison performed the first successful human vascular organ graft, a
kidney transplant [##REF##13278189##1##, ##REF##6371266##2##]. However, the donor
and recipient were monozygotic twins, obviating the need for immunosuppression
for organ graft survival. With the development of immunosuppressive regimens,
the same group 5 years later performed the first kidney allograft transplantation
between unrelated individuals; that graft survived for 20 years [##UREF##0##3##]. Although successful
graft survival was achieved, it rapidly became clear that all immunosuppressive drugs, even the newer
generations of immunosuppressive regimens, are toxic [##REF##16567244##4##, ##REF##17981639##5##]. Immunosuppressive drugs are also known to increase the risk of
infection and neoplasia [##REF##14662323##6##, ##REF##11833147##7##], and their associated side effects often lead to
patient noncompliance [##REF##11072057##8##]. Since most patients eventually reject transplanted allografts
either acutely or through a process of chronic rejection [##REF##7619392##9##–##REF##16418687##11##], these deleterious
side effects make organ transplantation a therapy in which the risk/benefit
ratio must be carefully weighed.</p> <p> To overcome issues associated with chronic
immunosuppression, investigators have focused on approaches that lead to the
induction of tolerance to transplanted organ allografts [##REF##9922369##12##]. Operationally, transplantation tolerance is defined as the
survival of a donor allogeneic graft in the absence of immunosuppression. Most
transplantation tolerance induction protocols take advantage of information
resulting from studies on the natural mechanisms by which the immune system prevents
self-reactivity and autoimmune disease. Two major forms of natural tolerance
have been identified: central tolerance and peripheral tolerance.</p>
<p>Recommended by Eiji Matsuura</p> <p>Transplantation of allogeneic organs has proven to be an effective therapeutic for a large variety of disease states, but the chronic immunosuppression that is required for organ allograft survival increases the risk for infection and neoplasia and has direct organ toxicity. The establishment of transplantation tolerance, which obviates the need for chronic immunosuppression, is the ultimate goal in the field of transplantation. Many experimental approaches have been developed in animal models that permit long-term allograft survival in the absence of chronic immunosuppression. These approaches function by inducing peripheral or central tolerance to the allograft. Emerging as some of the most promising approaches for the induction of tolerance are protocols based on costimulation blockade. However, as these protocols move into the clinic, there is recognition that little is known as to their safety and efficacy when confronted with environmental perturbants such as virus infection. In animal models, it has been reported that virus infection can prevent the induction of tolerance by costimulation blockade and, in at least one experimental protocol, can lead to significant morbidity and mortality. In this review, we discuss how viruses modulate the induction and maintenance of transplantation tolerance.</p>
<title>2. CENTRAL TRANSPLANTATION TOLERANCE</title> <p>In
1953, Peter Medawar et al. obtained the first experimental evidence that the
establishment of allogeneic hematopoietic chimerism leads to the induction of central
tolerance and permits permanent acceptance of skin allografts [##REF##13099277##13##]. Inspired by the work
done in freemartin cattle by Owen in 1945 [##REF##17755278##14##] and the clonal
selection theory subsequently proposed by Burnet and Fenner [##UREF##1##15##], Medawar demonstrated in
mice that the transfer of allogeneic hematopoietic cells in utero could induce
tolerance to skin transplanted from the original donor later in life [##REF##13099277##13##].</p> <p> Medawar's
observation led Main and Prehn to experimentally induce hematopoietic chimerism
by treating mice with whole-body irradiation (WBI) and allogeneic bone marrow
cells, followed by transplantation with donor-strain-matched skin allografts [##REF##13233946##16##]. This protocol successfully
induced tolerance to skin allografts, conclusively linking the establishment of
hematopoietic chimerism with subsequent allograft survival. However, despite
the long-term survival of skin allografts on mice treated with WBI and
allogeneic bone marrow, animals eventually develop lethal graft-versus-host
disease (GVHD), a reaction where passenger leukocytes in the donor bone marrow
or graft mount an immune response against the host. Therefore, modern conditioning
protocols to induce central tolerance have been designed to address the common
objectives of (1) establishing hematopoietic chimerism using a relatively
benign preconditioning protocol that (2) prevents the development of GVHD.</p> <p> Despite
these common objectives, modern conditioning regimens can differ quite
significantly in their methodology. In preclinical models of hematopoietic
chimerism, conditioning regimens span the spectrum from myeloablative
protocols, which often entail lethal irradiation and subsequent stem
cell rescue, to noncytoreductive treatments that do not require irradiation, for
example, costimulation blockade [##UREF##2##17##–##REF##10861026##19##]. Between these two
extremes are protocols that significantly weaken the recipient's immune system
through selective antibody-mediated elimination of specific immune populations (e.g., CD4<sup>+</sup> and CD8<sup>+</sup> T cells) coupled with targeted irradiation (e.g., thymic irradiation) [##REF##2562984##20##]. These latter protocols
are often considered nonmyeloablative. In clinical trials, successful
nonmyeloablative approaches have recently been described [##REF##18216356##21##, ##REF##18216355##22##]. Stable renal allograft
function in recipients for as long as five years after complete withdrawal of immunosuppressive
drugs was observed in recipients in which hematopoietic chimerism was
established [##REF##18216356##21##, ##REF##18216355##22##]. These reports
document that in humans, as in rodents, establishment of hematopoietic
chimerism is a robust approach for the development of central tolerance and the
permanent survival of donor-specific allografts.</p> <title>3. PERIPHERAL TRANSPLANTATION TOLERANCE</title> <p>The second major form of tolerance is peripheral
tolerance. Different from central tolerance in which hematopoietic chimerism
leads to the clonal deletion of antigen-specific cells during development,
peripheral tolerance targets pre-existing cells that have already been
generated. To induce tolerance in this population, fundamental insights into
how naive antigen-specific T cells become activated have led to protocols
designed to prevent this process. Naive T cell activation is initiated by the
interaction of the antigen-specific T cell receptor (TCR) with a peptide
presented by the MHC. This interaction conveys specificity leading to the
activation of only antigen-specific T cells. This signal is often termed as “signal
1” (##FIG##0##Figure 1##). Following
TCR-peptide/MHC ligation, a T cell then receives a number of costimulatory
signals [##REF##16611323##23##–##REF##16918529##25##]. A key costimulatory
signal in this pathway that permits the activated naive T cells to become
functional effector/memory T cells is provided by CD28-CD80/86 interaction [##UREF##3##26##], which has often been
referred to as “signal 2.” In early studies, it was shown in vitro that T cells that receive
signals through their TCR in the absence of engagement of the CD28-CD80/86
costimulation pathway became nonresponsive, a state of T cell nonresponsiveness
often referred to as anergy [##REF##9922369##12##, ##UREF##4##27##]. Following induction
of signal 2, cytokines are produced that impart the final signal for T cell
activation, and this is termed as “signal 3” [##REF##16824119##24##, ##REF##17513722##28##, ##REF##12732656##29##]. Although these three
critical signals are required for the full activation of T cells, additional
signals such as those derived from CD40-CD154 interaction can have potent
effects on the activation of naive T cells (##FIG##0##Figure 1##).</p> <p> The
existence of a comparable in vivo
state of T cell nonresponsiveness has been debated for years until it was independently
shown to exist by Ohashi et al. [##REF##1901764##30##] and Oldstone et al. [##REF##1901765##31##] using two very similar
experimental systems. These investigators generated double-transgenic mice that
expressed (1) lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) [##REF##1901764##30##] or nucleoprotein (NP) [##REF##1901765##31##] under the control of
the rat insulin promoter, and (2) a transgenic TCR that recognizes a peptide
from the transgenic LCMV protein. In unmanipulated mice, the transgenic T cells
migrate from the thymus into the peripheral tissues and encounter their cognate
antigen, but they remain nonresponsive to islets expressing GP or NP. However,
LCMV infection reverses this state of nonresponsiveness, leading to a diabetic
phenotype resulting from the destruction of pancreatic islets expressing the
transgenic protein [##REF##1901764##30##, ##REF##1901765##31##]. These data support a
mechanism where the LCMV-reactive T cells in naive mice encounter antigen in
the absence of costimulation and become nonresponsive (tolerant), and further
show that environmental perturbation can break this nonresponsive state. This model
serves as the conceptual basis for the induction of peripheral transplantation
tolerance, where the in vivo disruption
of the costimulatory process—referred to as
costimulation blockade—leads to the
induction of tolerance in an antigen-specific manner [##REF##9922369##12##].</p> <p>Costimulation
blockade therapies can target several different steps in the process of T cell
activation. However, the CD40-CD154 pathway linking signal 1 to signal 2 has been
identified to be a critical step in the activation of naive T cells. Anti-CD154
mAb blocks the interaction between CD154 on the T cell and CD40 on the APC [##REF##9368772##32##, ##UREF##5##33##], and prevents the differentiation
between naive T cells and effector/memory T cells [##UREF##5##33##] (##FIG##0##Figure 1##).</p> <p> In peripheral tolerance induction protocols,
anti-CD154 monotherapy significantly improves islet [##REF##7568172##34##] and cardiac [##REF##8560571##35##] allograft survival in
mice and islet allograft survival in nonhuman primates [##REF##11707732##36##–##REF##15816883##39##]. In combination with a
donor-specific transfusion (DST), anti-CD154 monoclonal antibody (mAb) induces
permanent islet [##REF##7568172##34##] and prolonged skin [##REF##9256196##40##] allograft survival in
mice. DST provides selective activation of the alloantigen-specific T cells,
and we have shown that the subsequent blockade of costimulation by anti-CD154
mAb leads to selective depletion of only the specific alloantigen-reactive CD8<sup>+</sup> T cells [##REF##11714833##41##, ##REF##10605049##42##]. Another reagent,
CTLA4-Ig, binds to the costimulatory molecules CD80/86 on the APC. This blocks
its interaction with CD28 on the T cell, preventing signal 2. CTLA4-Ig
monotherapy induces the survival of xenogeneic islets [##REF##1323143##43##] and allogeneic cardiac
grafts [##REF##8228826##44##]. Not surprisingly, the
combination of anti-CD154 mAb and CTLA4-Ig has shown great potential in prolonging
skin and cardiac allograft survival in mice [##REF##8632801##45##].</p> <p> Effective 
as a peripheral tolerance induction protocol, costimulation blockade protocols based
on blockade of the CD40-CD154 pathway have also been used to establish
hematopoietic chimerism leading to the generation of central tolerance [##UREF##2##17##–##REF##10861026##19##]. By establishing
multilineage hematopoietic chimerism, these noncytoreductive protocols have
proven to promote robust transplantation tolerance to a variety of solid-organ
allografts across fully allogeneic barriers when transplanted several weeks
after bone marrow transplantation (BMT) [##UREF##2##17##, ##REF##12952928##18##] or being concurrent
with BMT [##REF##10861026##19##, ##REF##17564635##46##]. Furthermore, because
donor-reactivity against the host is dependent on the CD40-CD154 pathway [##REF##7521888##47##], costimulation blockade
effectively establishes hematopoietic chimerism in the absence of GVHD [##UREF##2##17##, ##REF##12952928##18##].</p> <title>4. VIRUS INFECTION AND
TRANSPLANTATION TOLERANCE</title> <p> As costimulation blockade protocols move
closer to clinical reality, there is concern that virus infection during
tolerance induction may (1) induce tolerance to the virus, (2) prevent the
induction or maintenance of tolerance to the organ allograft, or (3) increase
risk to the host. Viruses are known to stimulate innate immunity by activating various
pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and retinoic
acid inducible gene-I- (RIG-I-) like receptors (RLRs) [##REF##17979849##48##]. Activation of innate
immunity by virus infection leads to the modulation of adaptive immunity, and it
has been shown to impair transplantation tolerance induction and allograft
survival [##REF##16869800##49##–##REF##10666251##57##].</p> <p>For example, infection with LCMV before [##REF##12682237##54##], at the time of [##REF##11673506##51##, ##REF##10945944##56##], or shortly after costimulation
blockade for the induction of peripheral tolerance [##REF##10666251##57##] impairs allograft
survival. Mice treated with costimulation blockade rapidly reject skin
allografts if they are infected with LCMV shortly after skin transplantation [##REF##10666251##57##]. Interestingly, this
effect appeared to be virus-specific, as infection with vaccinia virus (VV) and
murine cytomegalovirus (MCMV) did not engender allograft rejection [##REF##10666251##57##]. Furthermore, skin
allograft survival is significantly shortened in LCMV-immune mice treated with
a peripheral tolerance induction protocol consisting of DST and anti-CD154 mAb
combination therapies [##REF##12682237##54##]. Additionally, TLRs
and their proinflammatory role in responding to infection and ischemia are
being increasingly seen as a serious obstacle to solid-organ transplantation [##REF##16495791##58##–##REF##16785361##60##].</p> <p>Barriers to the induction of hematopoietic
chimerism and establishment of central tolerance in the setting of viral
infection have also been reported. Anti-CD154 mAb, CTLA4-Ig, and busulfan
treatment fails to induce bone marrow chimerism and tolerance to skin
allografts in the setting of multiple viral infections [##REF##12813024##53##]. Moreover, using a
nonmyeloablative protocol where anti-CD154 mAb treatment was coupled with
sublethal irradiation, Forman et al.
observed that infection with LCMV on the day of BM transplantation not only resulted
in allograft rejection but also proved lethal to the recipient [##REF##12055213##55##]. Interestingly,
conditioned recipients that were infected and given syngeneic BM grafts did not
die. Recipients of allogeneic BM died by a type I interferon- (IFN-) dependent
mechanism, whereas mice deficient in the type I IFN receptor survived. The
recent deaths of a cluster of human transplant recipients of LCMV-infected
organs make this finding particularly relevant to the safety and efficacy of
tolerance induction protocols based on costimulation blockade [##REF##16723615##61##, ##REF##18256387##62##].</p> <title>5. INNATE IMMUNE ACTIVATION BY VIRUS INFECTION</title> <p>It has
been shown that mice infected with LCMV concurrent to costimulation blockade
treatment [##REF##10945944##56##, ##REF##11064087##63##] or persistently infected with
LCMV clone 13 prior to costimulation blockade treatment [##REF##12421910##52##] rapidly reject skin
allografts. In a transgenic TCR model, LCMV prevents the deletion of
alloreactive CD8<sup>+</sup> T cells that is ordinarily induced by costimulation
blockade [##REF##10945944##56##, ##REF##11064087##63##]. In this same model system,
injection of a TLR agonist similarly prevents the deletion of host alloreactive
CD8<sup>+</sup> T cells which are required for skin allograft rejection [##REF##16424185##64##].</p> <p>Surprisingly,
the TLR4 agonist LPS impairs CD8<sup>+</sup> T cell deletion and shortens skin
allograft survival by activating host cells [##REF##16424185##64##] rather than donor cells [##REF##16424185##64##, ##REF##17982052##65##], even though the transgenic CD8
T cells recognize donor antigen via the direct pathway. Furthermore, LPS
required the expression of the adaptor molecule myeloid differentiation primary
response gene-88 (MyD88) on the recipient to shorten allograft survival [##REF##17982052##65##, ##REF##16970798##66##]. These findings are consistent
with clinical data suggesting that TLR4 polymorphisms on the host, but not the
donor, correlate with allograft survival [##REF##12773319##67##]. Together, these data suggest
that TLR activation induces a soluble mediator that augments host T cell
activation, perhaps through a process of bystander activation (see below).</p> <p>Numerous
cytokines are reported to be important in the activation of CD8<sup>+</sup> T
cells, including IL-12 [##REF##12732656##29##], TNF<italic>α</italic> [##REF##11739497##68##, ##REF##15383581##69##], and IFN-<italic>α</italic>/<italic>β</italic> [##REF##15814665##70##]. While IL-12 and TNF<italic>α</italic>
are dispensable for shortened allograft survival induced by LPS in
costimulation blockade treatment protocols [##REF##16424185##64##], IFN-<italic>α</italic>/<italic>β</italic> has
been reported to be absolutely essential for LPS to prime CTLs and induce
allograft rejection [##REF##17982052##65##]. Type I IFNs also proved
indispensable for allograft rejection mediated by the dsRNA mimetic and TLR3
agonist poly I:C [##REF##17982052##65##]. Emerging data suggest that
IFN-<italic>α</italic>/<italic>β</italic>
can be induced by viruses through a growing number of pathogen recognition
receptor systems [##REF##11607032##71##–##REF##12773480##74##].
Thus the induction of IFN-<italic>α</italic>/<italic>β</italic> by
virus infection or TLR ligation has emerged as an important obstacle to the establishment
of peripheral transplantation tolerance as well as to the maintenance of
self-tolerance [##REF##17562353##75##].</p> <title>6. SIGNALING PATHWAYS INVOLVED
IN INNATE IMMUNE CELL ACTIVATION
BY VIRUS INFECTION</title> <p> How
does virus-mediated activation of innate immunity lead to the production of IFN-<italic>α</italic>/<italic>β</italic>?
At present, the two best-characterized IFN-<italic>α</italic>/<italic>β</italic>-inducing
viral recognition systems are members of the TLR and the retinoic acid
inducible gene-I- (RIG-I-) like receptor (RLR) families (##FIG##1##Figure 2##). These receptors are activated by sensing viral nucleic
acids either in the cytosol (RLR) or in endosomes (TLR) of cells [##REF##17892846##76##]. Cytosolic receptors that
detect nucleic acids upon viral infection are expressed ubiquitously by
nucleated cells, while endosomal receptors, which detect viral particles that
are engulfed from outside rather than from direct infection, are expressed in
specialized cells of the innate immune system such as macrophages and dendritic
cells [##REF##16979569##77##].</p> <p>Cytosolic
RLRs, exemplified by the proteins RIG-I and melanoma differentiation factor-5
(MDA5), recognize double stranded RNA (dsRNA) located in the cytosol following
replication by an RNA virus, or infection by a dsRNA-genome virus, through
interaction with their helicase domains [##REF##17979849##48##]. RLRs contain a caspase
recruitment domain (CARD) [##REF##15208624##72##] which links detection of
viral dsRNA to transcription of IFN-<italic>α</italic>/<italic>β</italic> by forming homotypic interactions with
the CARD-containing molecule interferon-<italic>β</italic> promoter stimulator (IPS-1, also
known as mitochondrial antiviral signaling protein (MAVS), CARD adaptor
inducing IFN-B (CARDIF), and virus-induced signaling adaptor (VISA)) [##REF##16127453##79##–##REF##16153868##82##]. Activation of IPS-1 triggers
members of the I<italic>κ</italic>B kinase (IKK) family, specifically TANK-binding kinase 1
(TBK-1) and IKK<italic>ε</italic> (also known as inducible I<italic>κ</italic>B kinase, IKK-i), to phosphorylate
and activate interferon regulatory factory (IRF)-3 and/or IRF7 [##REF##16713980##83##–##REF##16306937##88##]. Once activated, IRF3 and
IRF7 translocate to the nucleus and bind to interferon-stimulated response
elements (ISREs) to induce the expression of IFN-<italic>α</italic> and IFN-<italic>β</italic>, as well as other
IFN-inducible genes [##REF##17979849##48##, ##REF##15800576##89##, ##UREF##6##90##].</p> <p> It has
recently been recognized that cytoplasmic sensing of DNA can also trigger IFN-<italic>α</italic>
and IFN-<italic>β</italic> production [##REF##16286919##91##–##REF##16301743##93##]. This pathway is thought to
intersect with the RIG-I and MDA5 pathways at the level of TBK-1 and IKK-I [##REF##16286919##91##], and it requires IRF3 for IFN-<italic>α</italic>/<italic>β</italic> induction [##REF##16413926##92##]. A candidate cytosolic recognition receptor that senses and is activated
by DNA has been described [##REF##17618271##94##]. This receptor, known as
DNA-dependent activator of IFN-regulatory factors (DAI), was reported to induce
type I IFN upon recognition of bacterial and mammalian as well as viral DNAs [##REF##17618271##94##].</p> <p>With
the exception of TLR4, all known TLRs that induce type I IFN recognize nucleic
acids, and are found in the endosomal compartment of cells. These include TLR3,
TLR7, TLR8, and TLR9. Unlike the cytoplasmic nucleic acid receptors, the
cellular distribution of endosomal TLRs is much more restricted. TLR7 and TLR9,
which recognize ssRNA [##REF##14976262##95##, ##REF##14976261##96##] and unmethylated DNA that
contain CpG motifs [##REF##11130078##97##], respectively, are expressed
highly on both conventional (cDC) and plasmacytoid (pDC) dendritic cells. However,
they can also be expressed on other hematopoietic cells, including B cells [##REF##15454922##98##, ##REF##14751761##99##]. TLR3, which recognizes dsRNA [##REF##11607032##71##], has a broader distribution
than TLR7 and TLR9, and can be found on nonhematopoietic cells such as
astrocytes and epithelial cells of the cervix, airway, uterus, vagina,
intestine, and cornea [##REF##17892846##76##, ##REF##15454922##98##–##REF##16497588##100##]. Its expression, however, is thought
to be highest in cDCs [##REF##17892846##76##, ##REF##16497588##100##].</p> <p>Similar
to the other non-IFN-<italic>α</italic>/<italic>β</italic>-inducing TLRs, TLR3, 7, 8, and 9 are capable of
activating both NF-<italic>κ</italic>B and MAPK cascades and triggering the transcription of
scores of proinflammatory cytokines and chemokines [##REF##17892846##76##, ##REF##14751761##99##, ##REF##16497588##100##]. However, the endosomal TLRs are
also capable of signaling through additional cascades, which results in the
expression of type I IFNs. Recognition of dsRNA by TLR3 results in the
activation of the adaptor molecule Toll/interleukin-1 receptor (TIR) domain-containing
adaptor protein inducing IFN-<italic>β</italic> (TRIF) [##REF##12539043##101##]. TRIF interacts with tumor
necrosis factor receptor-associated factor (TRAF)-3 to activate TBK1 [##REF##16306937##88##] and, as described above,
leads to the activation of IRF3 and IRF7 and induction of type I IFN. In
contrast, the coupling of TLR7 and TLR9 to IFN-<italic>α</italic>/<italic>β</italic> production involves the adaptor
molecule MyD88 [##REF##11130078##97##, ##REF##11812998##102##]. Following recognition of
either ssRNA or unmethylated DNA, a large complex consisting of MyD88, TRAF3,
TRAF6, IL-1 receptor-associated kinase (IRAK)-4, IRAK-1, IKK-<italic>α</italic>, and IRF-7 is
recruited to the TLR [##REF##17979849##48##, ##REF##16306936##87##, ##REF##16306937##88##, ##REF##15361868##103##–##REF##16612387##105##]. Following recruitment of the
complex, cytokines downstream of NF-<italic>κ</italic>B are stimulated, and type I IFN expression
is induced in an osteopontin (OPN) [##REF##16604075##106##] and IRF7-dependent fashion [##REF##17979849##48##, ##REF##15800576##89##]. Interestingly, stimulation
of TLR7 and TLR9 in cDCs is capable of inducing the expression of cytokines
that are downstream of the NF-<italic>κ</italic>B pathway, such as IL-6 and IL-12. However, only
pDCs are capable of producing IFN-<italic>α</italic> in response to ssRNA and CpG-containing DNA [##REF##17892846##76##]. As exemplified by the
multitude of signaling pathways by which TLRs can activate innate immunity, it
is clear that virus or microbial infection has multiple ways to active innate
immunity and modulate the adaptive immune system during tolerance induction.</p> <title>7. MECHANISMS OF VIRUS-MEDIATED MODULATION
OF TRANSPLANTATION TOLERANCE</title> <p>There
are multiple mechanisms by which virus infection or TLR agonists may modulate
tolerance induction and allograft survival. We will focus on three potential
mechanisms. First, virus infection can mature APCs to prime non-cross-reactive
T cells, a process called bystander activation [##REF##15621563##107##, ##REF##16418524##108##]. Second, virus infection may
stimulate innate immune cells to produce cytokines that suppress
tolerance-promoting regulatory T cells [##REF##16921386##109##]. Third, virus infection may
lead to the generation of virus-specific T cells that can cross-react with
alloantigens, a phenomenon known as heterologous immunity [##REF##16824126##110##].</p> <title>7.1. Bystander activation</title> <p> A
mechanism by which virus infection may modulate tolerance induction is through
bystander activation. As described above, virus infection activates innate
immunity, and is able to mature APCs to “license” them to activate non-cross-reactive
T cells. CD4<sup>+</sup> T cells are known to play a pivotal role in the
licensing of antigen-presenting cells (APCs) [##REF##14657225##111##]. The intercourse between
antigen-specific CD4<sup>+</sup> T cells and antigen-presenting APCs is thought
to be crucial for the generation of a full immune response. In the setting of
viral infection, virus-specific CD4<sup>+</sup> T cells facilitate the
maturation of virus-presenting APCs via CD154-CD40 interactions. Consequently,
the APC is stimulated to upregulate costimulatory molecules, as well as to secrete
proinflammatory cytokines. These molecules then feed back on the T cell,
stimulate it to become fully activated, and release additional inflammatory
cytokines and growth factors. Allospecific T cells that have encountered cognate alloantigen can be activated in this inflammatory milieu even if they do not cross-react with viral antigens. This process is traditionally referred to as bystander activation [##REF##14657225##111##].</p> <p>Viruses
have also been shown to mature APCs independently of the normally required CD154-CD40
interaction. LCMV infection stimulates the upregulation of MHC classes I and
II, CD40, CD80, and CD86 in the presence of CTLA-4-Ig and anti-CD154 mAb [##REF##11673506##51##]. The molecular mechanisms
that govern this process have not been fully elucidated; however, the induction
of type I IFNs by virus-infected APCs is a likely candidate. 
IFN-<italic>α</italic>/<italic>β</italic> is
known to directly induce the maturation of immature DCs, and it results in the
upregulation of MHC and costimulatory molecules [##REF##9712065##112##, ##UREF##7##113##]. Given that pDCs can produce
up to a thousand-fold more type I IFN than other cells [##UREF##7##113##, ##REF##14991609##114##], we propose that viral
detection by pDCs triggers the release of IFN-<italic>α</italic>/<italic>β</italic> that can in turn act in a paracrine
or autocrine fashion to mature alloantigen-presenting APCs (##FIG##2##Figure 3##). Thus,
these “IFN-<italic>α</italic>/<italic>β</italic>-licensed” alloantigen-presenting APCs could directly stimulate
alloreactive T cells, even in the presence of costimulation blockade.</p> <title>7.2. Regulatory cell suppression</title> <p>The induction and maintenance of CD4<sup>+</sup> regulatory T cells (Tregs) are essential
to allograft survival [##REF##9616216##115##–##REF##15371665##117##]. Therefore, a second
mechanism by which viruses could impair tolerance induction is through
modulation of the generation or activity of this important T cell subset. In addition
to priming cells through an IFN-<italic>α</italic>/<italic>β</italic>-dependent
mechanism, TLR activation also prevents the intragraft recruitment of regulatory
T cells in an MyD88-dependent manner [##REF##16970798##66##]. This observation extended
earlier work showing that the MyD88 pathway plays an important role in the
rejection of minor antigens [##REF##12750407##118##] and cardiac allografts [##REF##17015716##119##].</p> <p>IL-6 is
an MyD88-dependent cytokine that has emerged as a candidate mediator for
impairing regulatory T cell generation and function; its production is
diminished in untreated [##REF##17015716##119##]—as well as
LCMV-infected [##REF##15724245##120##]—mice deficient in
MyD88. CD4<sup>+</sup> T cells develop a FoxP3<sup>+</sup> regulatory T cell phenotype
when they are activated in the presence of TGF-<italic>β</italic>. However, when CD4<sup>+</sup> T cells are
activated in the presence of TGF-<italic>β</italic>
and IL-6, this regulatory phenotype is suppressed and the cells develop a
proinflammatory TH17 cell phenotype [##REF##16648838##121##] (##FIG##3##Figure 4##). Therefore, virus infection may precipitate allograft
rejection by preventing the generation of Tregs following costimulation
blockade and instead favor development of proinflammatory effector T cells.</p> <p>IL-6
has also been shown to be important in regulating antigen-specific adaptive
immune responses via additional mechanisms. Pasare et al. demonstrated that microbial induction of the TLR pathway
on DCs enabled effector T cells to overcome suppression by CD4<sup>+</sup>CD25<sup>+</sup> regulatory cells [##REF##12532024##122##] (##FIG##3##Figure 4##). They reported
that secretion of soluble mediators (principally IL-6) by TLR-activated DCs
could render effector T cells refractory to Treg-mediated regulation, permitting
activation of antigen-specific T cells in the presence of regulatory T cells. Hence,
virus infection may trigger allograft rejection by compromising key regulatory
mechanisms such as preventing the generation of regulatory T cells by
costimulation blockade as well as by enabling alloreactive T cells to escape
Treg-mediated suppression.</p> <title>7.3. Heterologous immunity</title> <p>The classic view of clonal T cell
activation is that one TCR interacts with one cognate antigen. However, we now
understand that TCR binding is degenerate, and can recognize multiple related
and unrelated antigens. The ability of an antigen-specific T cell to
cross-react with multiple antigens, known as heterologous immunity [##REF##16824126##110##], can influence
immunodominance, protective immunity, and immunopathology during subsequent
viral infections [##REF##16824126##110##, ##UREF##8##123##, ##REF##15528078##124##].</p> <p>In
studies of peripheral tolerance induction, of particular interest to transplant
scientists is the observation that virus-specific T cells cross-react with
alloantigens (##FIG##4##Figure 5##) [##REF##2418107##125##, ##REF##8093891##126##]. Yang et al. have reported that acute infection with VV, MCMV, or
arena viruses LCMV and pichinde virus (PV) resulted in the spontaneous
generation of cytotoxic lymphocytes (CTLs) with cytolytic activity towards
allogeneic cells [##REF##3487705##127##, ##REF##2537363##128##]. These results were further
supported by Nahill and Welsh [##REF##8093891##126##], who used limiting dilution
analyses to demonstrate that T cell clones specific for virus-infected
syngeneic cells also kill uninfected allogeneic targets. Our report using
virus-specific tetramers and an intracellular cytokine assay confirmed the
findings that LCMV infection led to the generation of virus-specific CD8 T
cells that cross-react with alloantigens, and further showed that virus-immune
mice were refractory to the induction of tolerance by costimulation blockade [##REF##10666251##57##]. Others have also reported
that virus-immune mice are refractory to tolerance induction by costimulation
blockade [##REF##12813024##53##]. Because memory T cells are
resistant to the induction of tolerance by costimulation blockade [##REF##15621563##107##, ##REF##16418524##108##], our data suggest that the
allo-cross-reactive virus-specific memory T cells may precipitate the rejection
of allografts even in the presence of costimulation blockade.</p> <title>8. VIRUS INFECTION AND ESTABLISHED
ALLOGRAFT SURVIVAL</title> <p>Surprisingly, mice infected with
LCMV one day after transplantation also exhibit shortened allograft survival [##REF##10666251##57##]. Interestingly, the longer time
after transplantation is, the less impact LCMV infection has on allograft
survival. The deletion of alloreactive CD8<sup>+</sup> T cells is thought to be
complete at this time [##REF##11714833##41##, ##REF##10605049##42##], making it improbable that
LCMV is interfering with deletion. However, because costimulation blockade
protocols are only implemented during the peritransplant period, it is possible
that LCMV infection shortly after transplantation prevents the generation of
regulatory T cells, which have been shown to require up to 30 days after
costimulation blockade to develop [##REF##11801641##129##]. Further research is
necessary to elucidate the mechanisms by which LCMV shortens allograft survival
during the post-transplantation timeframe.</p> <title>9. SUMMARY</title> <p>Viral infection presents a potent barrier to the induction of transplantation
tolerance. In this review, we have discussed potential mechanisms by which
viral infection modulates organ allograft survival in the setting of
transplantation tolerance. We have briefly summarized data on three mechanisms
by which viral infection may mediate these effects: bystander activation,
modulation of Tregs, or heterologous immunity. Recognition of viruses by
pattern recognition receptors on innate immune cells can also directly
stimulate the maturation of APCs, and thus may lead to bystander activation and
licensing of alloreactive T cells. Activation of APCs by viruses may trigger
the release of cytokines such as IL-6 that can prevent the generation and/or
function of regulatory T cells that are essential for transplantation
tolerance. Finally, heterologous immunity may be responsible for the
discrepancy that has been encountered when tolerance strategies that work in specific
pathogen-free rodent models fail when translated to nonhuman primates and to humans [##REF##16861933##130##], which have been exposed to a
variety of pathogens and thus have large memory T cell pools. Understanding the 
cellular and molecular mechanisms by which viruses and other microbial
organisms modulate transplantation tolerance may lead to novel approaches that
improve the efficacy of allogeneic organ transplantation.</p>
<title>ACKNOWLEDGMENTS</title> <p>This 
work is supported in part by the National Institutes of Health Research Grant
no. AI42669, the American Diabetes Association Grant no. 7-05-PST-02, the
Juvenile Diabetes Research Foundation, and a Diabetes Endocrinology Center
Research Grant
DK32520 from the National Institutes of Health. The contents of this
publication are solely the responsibility of the authors and do not necessarily
represent the official views of the National Institutes of Health. D. M. Miller
and T. B. Thornley contributed equally to this work.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>
<italic>Costimulation blockade</italic>. Activation of a T cell involves a series of interactive steps with an APC. The first signal imparts antigen specificity and commences when the TCR engages the antigen/MHC complex presented by the APC. This signal is commonly referred to as “signal 1.” In subsequent steps, the T cell receives a number of costimulatory signals, including those following interaction of CD154 on T cells with CD40 on APCs, which matures the APC to upregulate expression of CD80/86. The interaction of CD28 with CD80/86 is termed as “signal 2” and activates APCs to secrete cytokines, which provide the final activation signals to the T cell; this step is commonly referred to as “signal 3.” Protocols based on costimulation blockade can prevent T cell activation by targeting steps in the T cell activation cascade. Anti-CD154 mAb blocks the interaction between CD154 and CD40, and prevents the APC from upregulating CD80/86, blocking full APC activation. This prevents the secretion of proinflammatory cytokines, thus depriving the T cell of signal 3. As a result of costimulation blockade, the T cell does not develop an activated phenotype, and consequently becomes nonresponsive (tolerant) to allogeneic antigens.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>
<italic>Pathogen recognition systems</italic>. The innate immune system senses viral pathogens by recognizing distinct pathogen-associated molecular patterns (PAMPs) using various pattern recognition receptors (PRRs). Two of the best-characterized virus-sensing PRRs include member of the Toll-like receptors (TLRs) and retinoic acid inducible gene-I- (RIG-I-) like receptors (RLRs) families. These PRRs couple the recognition of viral PAMPs to the induction of proinflammatory cytokines through various signaling cascades. The cytosolic RNA helicase receptors MDA5 and RIG-I initiate the cascade by recruiting the Cardif/TBK1 complex after sensing viral RNA. This activates the kinase TBK1 to phosphorylate interferon regulatory factor (IRF)-3 and IRF7, resulting in their nuclear translocation and the transcription of IFN<italic>α</italic>/<italic>β</italic>. The cell surface receptor TLR4, in partnership with CD14, couples the recognition of respiratory syncytial virus fusion protein [##REF##11062499##78##] to cytokine induction by signaling through the MyD88-dependent as well as the MyD88-independent pathways. The endosomal TLRs, TLR7, TLR8, and TLR9 also signal through MyD88 to activate inflammatory cytokines such as TNF, IL-6, and IFN-<italic>α</italic>/<italic>β</italic>. The other endosomal TLR (TLR3) signals through the MyD88-independent pathway via the TIR domain-containing adaptor molecule TRIF. Via TRIF, TLR3 signaling can activate NF-kB using TRAF6, and in addition, can induce type I IFN expression probably via TRAF3, TBK1, and IRF3.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>
<italic>Bystander activation of alloreactive T cells by “virus-licensed” APCs</italic>. Upon viral infection, detection of pathogen-associated molecular patterns by PRRs can stimulate the production of inflammatory cytokines such as IFN-<italic>α</italic>/<italic>β</italic>, TNF-<italic>α</italic>, and IL-6. These cytokines activate alloantigen-processing APCs in a paracrine or autocrine fashion to upregulate MHC classes I and II, as well as costimulatory molecules, such as CD80 and CD86. The heightened expression of costimulatory molecules elicits the proliferation and differentiation of alloreactive T cells.</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>
<italic>Modulation of regulatory mechanisms by virus infection</italic>. Regulatory T cells play a crucial role in transplantation tolerance to allogeneic organs. Regulatory mechanisms that prevent immune attack on allogeneic tissues may be compromised in the setting of viral infection by at least two mechanisms. Release of inflammatory cytokines by virus-infected cells can prevent the differentiation of uncommitted naive CD4<sup>+</sup> T cells into Tregs. Naive CD4<sup>+</sup> T cells can differentiate into regulatory T cells in the presence of TGF-<italic>β</italic>. However, in the presence of TGF-<italic>β</italic> and proinflammatory cytokines such as IL-6, and perhaps IL-21, naive T cells can be skewed to turn into effector T cells such as the IL-17-producing TH17 cells. In a separate mechanism, release of cytokines such as IL-6 by infected APCs can render alloreactive effector cells refractory to suppression by regulatory T cells.</p></caption></fig> <fig id="fig5" position="float"><label>Figure 5</label><caption><p>
<italic>Heterologous immunity; cross-reactivity between viral and allogeneic antigens.</italic> Unlike the very small proportion of naive T cells that can respond to any given pathogen (reported to be ~1:200 000), the frequency of T cells that directly recognize allogeneic antigens, such as MHC, is thought to be within 1:100–1:10. A proportion of those TCRs that recognize alloantigens, therefore, may have arisen as a result of virus infection that induces virus-specific T cells that cross-react with allo-MHC. Activation of these T cells may result in the recognition of MHC molecules found on donor tissues, such as the endothelium of transplanted organs, precipitating allograft rejection.</p></caption></fig>
<graphic xlink:href="CDI2008-742810.001"/> <graphic xlink:href="CDI2008-742810.002"/> <graphic xlink:href="CDI2008-742810.003"/> <graphic xlink:href="CDI2008-742810.004"/> <graphic xlink:href="CDI2008-742810.005"/>
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Clin Dev Immunol. 2008 Sep 14; 2008:742810