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CONCISE REVIEW |
Department of Oncology, Immunology and Hematopoiesis Division, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, USA
Key Words. Fas ligand (FasL) • Immune privilege • Allogeneic • Tolerance • gld • lpr • Dendritic cells
Correspondence: Curt I. Civin, M.D., Sidney Kimmel Comprehensive Cancer at Johns Hopkins, Buntin-Blaustein Cancer Research Bldg., Room 2M44, 1650 Orleans Street, Baltimore, MD 21231 USA. Telephone: 410-955-8816; Fax: 410-955-8897; e-mail: civincu{at}jhmi.edu
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ABSTRACT |
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 Top Abstract IntroductionFasL In Tissue And...FasL And Autoimmune DiseasesFasL-Transduced Dcs For...Limitations of Fasl UsageSummaryReferences |
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INTRODUCTION |
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TopAbstractIntroductionFasL In Tissue And...FasL And Autoimmune DiseasesFasL-Transduced Dcs For...Limitations of Fasl UsageSummaryReferences |
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Mature peripheral T lymphocytes undergo rapid expansion after encountering antigen. To restore homeostasis and avoid autoimmunity, the majority of the antigen-stimulated activated T cells are eliminated. This process, termed "peripheral deletion," involves Fas/FasL-mediated apoptosis [10–12]. In lymphoid organs, coexpression of Fas/FasL on activated T cells can lead to activation-induced cell death (AICD). In the periphery, infiltrating activated T cells induce upregulation of FasL on nonlymphoid tissues and thereby indirectly mediate their own apoptosis [13–15]. Fas/FasL interactions have also been implicated in CD8+ cytotoxic T lymphocytes (CTLs) [16–18], natural killer (NK) cells [19,20], and CD4+ CTLs [21–23].
Both lpr and gld mice express high autoantibody titers. Several studies demonstrated the importance of Fas/FasL interactions in the elimination of autoreactive B cells in normal mice. Rathmell and colleagues [24] used the hen egg lysozyme (HEL) model antigen and showed that only Fas-expressing autoreactive anti-HEL B cells were eliminated by HEL-specific CD4+ T cells. Wang and Shlomchik [25] showed that, in Fas-deficient mice, autoreactive B cells with rheumatoid factor specificity are activated in the presence of the autoantigen, while they are ignored in normal mice. In another study, Melamed et al. [26] demonstrated the importance of Fas in the elimination of defective B cells during development and prevention of autoimmunity.
FasL (CD95L, Apo-1L, CD178) is a type 2 conserved membrane protein of 280 amino acids (~40 kDa) that belongs to the tumor necrosis factor (TNF) family. This family also includes the death-receptor TNF
, TNF-related apoptosis-inducing ligand (TRAIL/Apo-2L), and TNF weak inducer of apoptosis (TWEAK/Apo-3L) [27]. FasL binding to its cognate receptor Fas (CD95, Apo-1) triggers the extrinsic apoptotic pathway, leading to the formation of the death-inducing signaling complex (DISC) as a consequence of recruitment of the adaptor protein FADD (Fas-associated death domain) to the Fas receptor intracellular death domain and the binding of FADD to procaspase-8 (FLICE) or -10 (FLICE-2) [28–35]. Binding of procaspase-8 to FADD leads to its autocleavage and activation. Scaffidi et al. [36] and Barnhart et al. [37] proposed that the activation of caspase-8 could lead to cell death via two different pathways, depending on the amount of active caspase-8 that is produced at the DISC. If a high amount of activated caspase-8 is produced, which is characteristic of "type 1 cells," caspase-8 can directly initiate a caspase cascade, starting with the direct cleavage and activation of the effector procaspase-3, which then leads to cleavage of many proteins and cell death. Anti-apoptotic proteins such as Bcl-2 and Bcl-xL cannot protect type 1 cells from the death receptor–induced cell death. If a low amount of activated caspase-8 is produced, which is characteristic of "type 2 cells," Fas/FasL-mediated apoptosis relies completely on the mito-chondrial amplification loop, the intrinsic pathway, which starts with the cleavage of the pro-apoptotic Bcl-2 family member Bid by caspase-8 [38,39]. Truncated Bid enters the mitochondria and initiates the release of cytochrome c and mitochondrial dysfunction by activating the pro-apoptotic Bcl-2 family members Bak and Bax [40,41]. Cytochrome c triggers the formation of the cytochrome c/Apaf-1/caspase-9 complex, termed the "apoptosome," which activates caspase-9 to subsequently cleave the effector caspases, caspase-3 and caspase-7 [42,43]. Fas-mediated cell death in type 2 cells can be significantly inhibited by Bcl-2 to Bcl-xL [36]. In addition, type 2 cells taken from Bid-deficient and Bax/Bak double deficient mice are resistant to Fas-mediated cell death [44,45]. The Fas/FasL apoptotic pathway is highly regulated (by regulatory mechanisms that are specific for Fas and others common to death receptors), and its abnormal regulation has been associated with cancer and autoimmunity [46].
Membrane FasL (mFasL) can be cleaved by matrix met-alloproteinases (MMPs) at a conserved cleavage site into a 26-kDa trimeric soluble form of FasL (sFasL), which consists of the FasL extracellular region [47,48]. There is some controversy regarding the ability of sFasL to induce apoptosis. In part, this controversy might be due to the use of different forms of sFasL. To address the role of sFasL in mediating apoptosis in vivo, mice deficient in matrilysin (MMP-7), one of the MMPs that generates sFasL, were studied. These mice showed reduced Fas/FasL-mediated apoptosis of epithelial cells during prostate involution [49]. Using supernatants of FasL-expressing cells as a source for the native sFasL, Aoki et al. [50] showed that the cytotoxic activity of this sFasL is increased after its binding to extracellular matrix proteins. These findings led Aoki et al. to suggest that sFasL binding to extracellular matrices might be important for immune tolerance. Findings showing that the level of serum sFasL is correlated with disease progression of several malignancies [51–53] and that tumor cells can evade immune surveillance by secreting sFasL to induce apoptosis of attacking T cells [54,55] support the suggestion that native sFasL is active in inducing apoptosis.
Unlike Fas, which is constitutively expressed by various cell types, the tissue distribution of FasL is limited. FasL is predominantly expressed on activated T lymphocytes [56] and NK cells [19]. It is also expressed at immunologically privileged sites, such as the eye [57], brain [58,59], lung [60], placenta and pregnant uterus [61], where the inflammatory response is physiologically limited. One of the mechanisms to protect organs in immune-privileged sites is constitutive or induced expression of FasL [62]. For instance, FasL protects the eye by mediating apoptosis of Fas+ lymphoid cells entering this organ in response to viral infection; in gld mice (with mutated FasL), the eye is not protected from inflammatory damage [57]. This phenomenon was broadened by Hu et al. [63], who showed that mice with either mutated Fas or mutated FasL have a greater intensity of toxoplasma-induced intraocular inflammation than do wild-type mice.
The ability of FasL to protect tissues from immune damage is further demonstrated in corneal transplantation. Corneal transplants are the second most-used form of tissue transplant, and a high degree of graft acceptance is achieved without tissue matching. The protective role of FasL was demonstrated by showing that the constitutive expression of FasL on the cornea is important for its graft acceptance. FasL– corneas (from gld mice) were never accepted [64].
FasL expression has also been observed in different tumors such as colorectal carcinoma [65–68], melanoma [69,70], head and neck carcinomas [71,72], hepatocellular carcinoma [73], lung carcinoma [74,75], and myeloma [76]. Studies in vitro showed that FasL+ tumor cells mediate the apoptosis of Fas-sensitive lymphoid cells [65, 71, 73, 74, 76, 77]. Other studies showed that melanoma cells expressing FasL had delayed tumor growth in lpr mice [70] and that in FasL+ esophageal and colorectal tumor regions, fewer tumor-infiltrating lymphocytes but more apoptotic lymphocytes were detected, in comparison to FasL– regions of these tumors [66,78]. Based on these and other findings, it has been suggested that FasL-expressing tumor cells are immune privileged and that FasL expression is one of the mechanisms used by cancer cells to evade immune surveillance. This hypothesis was challenged by studies showing that enforced over expression of FasL in FasL– tumors led to their rejection [79,80]. As will be discussed later, controlled FasL expression and its coexpression with other proteins, such as transforming growth factor-beta (TGF-ß), that determine the ability of FasL to mediate immune privilege, are components of strategies to achieve the benefits of FasL as a protective molecule.
The role of FasL in immune-privileged sites and its ability to protect various tumors led researchers to hypothesize that expression of FasL on allogeneic tissue transplants would protect the allograft from immune rejection. In this review, we present the findings of using FasL to reduce allograft rejection. We also present the findings of using FasL to treat different autoimmune diseases and the use of FasL-transduced dendritic cells (DCs) to achieve antigen-specific immuno-suppression and peripheral tolerance. We discuss the problems with this method and suggest ways to solve them.
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FASL IN TISSUE AND ORGAN TRANSPLANTATION |
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Immunosuppressive drugs that nonspecifically inhibit the immune system are the current treatment mainstays to prevent allograft rejection. This treatment does not absolutely exclude the possibility of chronic rejection [82]. In addition to the direct toxic side effects of some immunosuppressants, patients treated with these drugs have increased risk of life-threatening infections and malignancies [83–85]. Based on the protective role of FasL in immune-privileged sites and tumors, and its importance for the acceptance of corneal allografts, different groups analyzed the ability of FasL to protect allogeneic tissues and organs as an alternative to immunosuppressive drugs. FasL-mediated apoptosis of lymphocytes in immune-privileged sites not only protects the sites from immune damage but also induces immune tolerance. Griffith et al. [86] showed that FasL-mediated apoptosis of lymphocytes invading a virally infected eye led to induction of tolerance to the specific virus; such tolerance was not induced in gld and lpr mice. Kawashima et al. [87] supported these findings using allogeneic splenocytes instead of virus. The importance of T-cell apoptosis for the induction of tolerance was demonstrated in several additional studies (some of these demonstrated the importance of Fas/FasL-mediated apoptosis) [88–93]. It was suggested that the mechanism underlying the ability of apoptotic cells to induce tolerance in the eye is promotion of Th2 response mediated by APCs that engulfed interleukin (IL-10)–producing apoptotic cells [94]. The attempts to protect and induce tolerance to different allogeneic tissues and organs by using FasL are presented below.
Kidney
An initial attempt by Swenson et al. [95] to examine the ability of FasL to protect allogeneic kidneys from rejection showed that, although adenoviral-FasL–transduced allogeneic rat kidneys expressed FasL for only approximately 2 weeks, the mean survival of animals transplanted with FasL+ renal allografts was 28 days, compared with control animals with survival of 12 days. In another study from the same group, Ke et al. [96] showed that animals transplanted with FasL+ renal allografts survived until the experiment was ended at day 56 post-transplantation, while the control animals survived only 12 days. The extended period of survival was correlated with the persistently increased expression of FasL in the transplanted tissue throughout the observation period. Prolonged survival of animals transplanted with FasL+ kidneys was correlated with downregulation of the anti-apoptotic protein Bag-1 and with a shift toward Th2 response at the graft site.
Heart and Blood Vessels
An early study by Takeuchi et al. [97] showed that FasL-expressing hearts from FasL transgenic mice, when transplanted into the abdomen of isogeneic or allogeneic mice, underwent accelerated rejection (after ~1.5 days) accompanied by leukocyte infiltration; in this study nontransgenic isografts were also rejected (after 21 days).
In contrast, Askenasy and colleagues [98] demonstrated the protective effect of FasL on heart transplants using a different approach. They displayed high quantity of FasL on the murine-transplanted heart vasculature by binding a chimeric streptavidin-FasL protein (comprised of the extracellular region of FasL attached to streptavidin) to biotin-modified hearts. The presence of FasL on the cardiac endothelium did not affect cardiac function and was not toxic to the heart or liver. Despite the short half-life of the chimeric streptavidin-FasL protein (9 days) in vivo, allogeneic hearts displaying streptavidin-FasL had a prolonged survival, compared with the control hearts displaying a chimeric streptavidin-FasL mutant protein in which FasL was inactive. Further prolongation of graft survival was achieved by post-transplant injection of a single dose of donor splenocytes decorated with streptavidin-FasL. This study highlights an important consideration that should be taken into account in transplantation of vascularized organs such as the heart. In these, in order to protect the allograft from immune rejection, FasL should be expressed not only on the organ-specific cells but also, and more important, on the organ blood vessels, which are the first line of defense and may block the penetration of inflammatory destructive cells to the tissue.
Instead of expressing FasL on the transplanted tissue, Min et al. [99] used FasL-transduced donor DCs as another approach to induce donor-specific transplantation tolerance (for more information, see the DC section below). Min et al. showed that three sequential injections of donor DCs transduced with FasL administered before transplantation of the donor allogeneic hearts led to prolonged allograft survival, compared with control mice injected with donor DCs transduced with an empty control vector. Induction of alloreactive T-cell apoptosis by the donor-transduced DCs was shown to be the underlying mechanism.
The ability of FasL overexpressed on the vascular endothelium to limit the inflammatory response was demonstrated in several other studies. FasL is normally expressed on the vascular endothelium, and it was shown that downregulation of FasL expression by the pro-inflammatory cytokine TNF-induced mononuclear cell infiltration and that overexpression of FasL-attenuated TNF-induced leukocyte infiltration [100]. This important finding led Sata et al. [101] to suggest that overexpression of FasL on vascular endothelium can be used to prevent transplant arteriosclerosis. Sata and his colleagues [101] showed that overexpression of FasL on the endothelium of rat donor carotid arteries significantly inhibited transplant-associated intimal hyperplasia and that the inhibition of neointima formation was accompanied by a reduction in T-lymphocyte and macrophage infiltration of the grafted vessels. Intimal hyperplasia generated by balloon injury to the carotid arteries could also be inhibited by delivery of adenoviral FasL to the injured site in rats [102,103]. In a study that was aimed to inhibit the inflammatory reaction that contributes to the pathogenesis of myocardial ischemia-reperfusion injury, Yang et al. [104] created transgenic mice that overexpress human FasL on vascular endothelium and showed that, following ischemia-reperfusion injury, the transgenic mice had significantly reduced infarct size and improved myocardial function compared to nontransgenic mice. The reduction in myocardial damage was attributed to the significant reduction in neutrophil accumulation in the re-perfused tissue of the transgenic mice.
Liver
Li et al. [105] showed that the ability of FasL to protect liver allografts depended on the expression level of FasL. Rats transplanted with FasL-allogeneic livers (in which ~10% of the cells expressed FasL) survived significantly longer than the control group. Apoptosis of infiltrating lymphocytes was observed in the FasL-transfected livers. In contrast, rats transfected with higher amounts of FasL DNA developed lethal hepatitis.
Lung
Schmid et al. [106] showed that rat lungs, efficiently transduced with FasL using liposomes, in combination with a low dose of the immunosuppressant cyclosporin A, had significantly better gas exchange than did a control group receiving liposomes that did not contain the FasL vector or cyclosporine A. Histologic analysis of the FasL-transduced grafts showed clusters of apoptotic cells that were not seen in the untreated grafts; compared with the untreated grafts, lymphocyte infiltrates were less extensive in FasL-expressing grafts.
Skin
Expression of FasL on skin allografts has also been shown to be protective. Saitoh et al. [107] found that retinoic acid (RA) induces FasL expression on dermal fibroblasts, and they showed that daily injection of mice with RA under a skin allograft prolonged their survival (12 days), compared with control allografts injected with a vehicle control (5 days). When the donor allograft was from a gld mouse (mutated FasL), allograft survival was not changed by treatment with RA. Likewise, anti-FasL antibodies injected together with RA could partially abrogate the protective effect of RA.
Thyroid
Tourneur et al. [108] showed that the ability of FasL, which had been transgenicly expressed on thyroid follicular cells, to prevent thyroid allograft rejection depended on its expression level. Thyroids expressing high levels of FasL had prolonged survival (42 days) compared to thyroids expressing low levels of FasL and control thyroids (7–14 days). In contrast, when transplanted into lpr mice, thyroids expressing high levels of FasL were rejected. The prolonged survival of allogeneic thyroids expressing high levels of FasL was attributed to the decrease in infiltrating lymphocytes, inhibition of the allospecific cytotoxic T-cell response, and a shift toward a Th2-antibody response. Also, CD4+ T cells were shown to be involved in FasL-induced allograft protection, since Fas+ thyroids were rejected from mice depleted of CD4+ T cells.
Pancreatic Islet ßcells
The first attempts to confer protection to allogeneic pancreatic islets by expression of FasL failed [109,110]. It is important to note that, in these studies, the expression of FasL in the pancreatic ßcells of the transgenic mice led to their destruction. To overcome the possible FasL sensitivity of pancreatic ßcells, pancreatic islets were co-transplated with FasL-expressing passenger cells. Testicular cells naturally expressing FasL (Sertoli or sperm cells [111]) were able to protect pancreatic islets taken from the same mice donor when co-transplanted to diabetic allogeneic–recipient mice [112,113]. Donor pancreatic islets co-transplanted with donor testicular cells had prolonged survival (17.5 ± 9.1 days) compared to the control islets co-transplanted with gld donor testicular cells (10.4 ± 2.6 days), in chemically diabetic allogeneic mice; no prolongation of survival was observed in lpr recipients (9.8 ± 3.4 days) or in recipient mice treated with anti-FasL antibodies (11.8 ± 2 days) [113]. The ability of FasL-expressing cells to protect allogeneic pancreatic islets was also demonstrated by Lau et al. [114]. Syngeneic myoblasts genetically engineered to express FasL protected donor pancreatic islets from immune rejection in diabetic allogeneic mice and restored normal glucose levels in the blood. In contrast to Lau et al., Turvey et al. [115] and Kang et al. [116] found that FasL-expressing skeletal myoblasts did not protect either syngeneic or allogeneic pancreatic islets respectively from immune destruction.
In another study, using an approach similar to that used by Askenasy et al. [98],Yolcu et al. [117] displayed a high quantity of FasL on allogeneic donor mouse splenocytes by attaching a chimeric streptavidin-FasL protein to biotin-modified cells and analyzed their ability to protect donor pancreatic ßcells. Co-transplantation of donor pancreatic ßcells with the donor streptavidin-FasL–decorated splenocytes to diabetic allogeneic mice prevented the rejection of the ßislets (during the 56-day observation period) and restored glucose home-ostasis. In contrast, pancreatic ßcells co-transplanted with streptavidin–mutant FasL (inactive control) were rejected by 8 days. The ability of streptavidin-FasL splenocytes to block primary and secondary alloreactive responses in vivo and the finding that this blocking depended on Fas expression led the authors to suggest that streptavidin-FasL splenocytes protect allogeneic pancreatic islets by inducing Fas/FasL-mediated apoptosis of alloreactive lymphocytes.
Bone Marrow
Allogeneic bone marrow transplantation (allo-BMT) is an important therapeutic modality for hematological malignancies and hematopoietic disorders. Graft-versus-host disease (GVHD), which is mediated by T lymphocytes present in donor bone marrow, is the primary contributing factor to morbidity and mortality after allo-BMT [118]. Depletion of donor T lymphocytes reduces both the incidence and the severity of GVHD, but it is also associated with increased rates of graft rejection, impairment in immune reconstitution, and increased incidence of cancer relapse [119]. To avoid the consequences of depletion of all T cells from the donor bone marrow, several strategies have been developed, including depletion of residual host T cells [120]; depletion of selective T-cell subsets from the donor bone marrow, such as mature CD6+ T cells and TCR- ßT cells [121–123]; and escalation of CD34+ stem-progenitor cell allograft dose [124,125]. Looking for a new approach to prevent graft rejection, Whartenby et al. [126] examined the ability of FasL to protect transplanted allogeneic stem cell–enriched bone marrow cells from acute rejection after nonmyeloablative transplantation. Significant enhancement in short-term engraftment in a multiple minor histocompatibility–mismatch mouse model was achieved with FasL-transduced donor cells, compared with control allografts transduced with the empty vector, even though their efficiency of FasL transduction was only 10%–20%. No immune impairment, liver toxicity, or reduction in marrow function was observed in mice transplanted with allogeneic FasL-transduced donor allografts.
Allo-BMT has also been shown to be important for induction of tolerance to solid organs provided by the same donor who provides the marrow allograft [127–129]. George et al. [130] used a mouse model of antithymocyte globin and donor bone marrow to examine the involvement of Fas/FasL in bone marrow–induced transplantation tolerance. They showed that promotion of skin allograft acceptance depended on FasL expression on the skin donor’s bone marrow cells, since bone marrow from gld donors did not prolong skin allograft survival. Goldstein et al. [131] used the same mouse model and showed that Fas expression on the recipient cells is also important for bone marrow–induced transplantation tolerance. These findings led the authors to suggest that donor bone marrow induces transplantation tolerance by deletion of recipient allograft-reactive cells via Fas/FasL-mediated apoptosis.
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FASL AND AUTOIMMUNE DISEASES |
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TopAbstractIntroductionFasL In Tissue And...FasL And Autoimmune DiseasesFasL-Transduced Dcs For...Limitations of Fasl UsageSummaryReferences |
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Hashimoto’s disease is the most common type of autoimmune thyroiditis; autoreactive CD4+ T lymphocytes initiate an autoimmune response that leads to the destruction of thyroid cells and clinical hypothyroidism [132]. Batteux et al. [133] examined the ability of FasL to protect the thyroid gland from autoimmune destruction in a murine model of Hashimoto’s thyroiditis that was induced by immunization with mouse thyroglobulin. The ability of transgenic mice expressing FasL on thyroid cells to prevent the induction of experimentally induced thyroiditis depended on the level of FasL expression. Mice expressing high levels of FasL showed minimal inflammatory infiltration of the thyroid, diminished autoreactive CTL and CD4+ T proliferative responses, and decreased autoantibody and Th1 cytokine production compared with control mice and mice expressing low levels of FasL.
Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system (CNS) in which T cells reactive with proteins of the myelin sheath lead to nerve immune injury and permanent disability [134]. Experimental autoimmune encephalomyelitis (EAE), the animal model for MS, can be induced by immunization of animals with myelin proteins such as myelin basic protein (MBP) and pro-teolipid protein (PLP) or by transferring activated T cells, specific for myelin proteins, to the host animal [135]. Zhu et al. [136] demonstrated the ability of FasL to prevent the development of MBP-induced EAE in Lewis rats. Rats that underwent intrathecal infusion of recombinant FasL had reduced infiltration of inflammatory cells (T cells and macrophages) into the lumbosacral spinal cord and, consistent with this, an increased percentage of apoptotic inflammatory cells compared with the control rats. This treatment did not damage the myelin sheath or neural cells in the spinal cord. In vitro, it was shown that both MBP-specific encephal-itogenic T cells and activated macrophages are sensitive to recombinant FasL-induced cell death. Systemic administration of recombinant FasL could not prevent EAE, showing that FasL acted locally within the CNS to prevent EAE. In other studies of EAE models in lpr and gld mice, Fas/FasL-mediated apoptosis has been shown to be important for the recovery from EAE [58,59].
Rheumatoid arthritis is a chronic, progressive, systemic autoimmune disease that results in inflammation and destruction of synovial joints and often erosion of the adjacent cartilage and bone, leading to substantial disability [137]. The animal model for rheumatoid arthritis, collagen type II (CII)–induced arthritis, involves both T- and B-cell immunity to CII for disease manifestation [138]. Zhang et al. [139] used the CII-induced arthritis model in DBA/1 mice and showed that administration of four boosts of FasL-transduced CII-macrophages, 2 weeks after the first immunization and 2 weeks before the second immunization of the mice with CII, could significantly inhibit the development of rheumatoid arthritis, compared with the control mice treated with green fluorescent protein (GFP)–transduced CII macrophages. The treatment with FasL-transduced CII macrophages led to the elimination of CII-specific T cells, without impairment of the host immune response to other antigens such as ovalbumin (OVA). This study shows that, in order to achieve complete inhibition of development of rheumatoid arthritis, both deletion of CII-activated cells and blocking of B-cell activation are needed and that T cells have a dominant role in CII induction of rheumatoid arthritis. In another study, using lpr and gld mice, Hsu et al. [140] demonstrated the importance of T-cell apoptosis via Fas/FasL for the ability to prevent the development of chronic arthritis subsequent to mycoplasma infection.
Autoimmune myasthenia gravis (MG) is characterized by weakness of skeletal muscles as a result of impaired neuromuscular transmission caused by antibody-mediated damage to acetylcholine receptors [141]. Wu et al. [142,143] demonstrated the ability of APCs engineered to express both acetylcholine receptor (AchR) and FasL to eliminate AchR-specific T cells. AchR-specific T cells from lpr mice were not eliminated, showing the involvement of the Fas/FasL-apoptotic pathway in T-cell depletion [144]. Preliminary studies in vivo demonstrated the immunotherapeutic potential of this strategy for MG [144].
Sjogren’s syndrome (SS) is a chronic autoimmune disease that is characterized by destruction of the exocrine salivary and lacrimal glands by invading lymphocytes, especially T cells [145]. Infection of lpr and gld mice with murine cytomegalovirus (MCMV) leads to the development of a chronic sialadenitis with severe salivary gland inflammation similar to SS [146,147]. Fleck et al. [147] used the SS model in B6-gld/gld mice and showed that local injection of the FasL gene, using the Cre/LoxP system, to the salivary glands at day 14 (acute sialadenitis) and day 75 (chronic sialadenitis) post MCMV infection led to significant reductions in the size and number of inflammatory foci, compared with the control. The treatment itself was not toxic to the gld mice.
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FASL-TRANSDUCED DCS FOR INDUCTION OF PERIPHERAL TOLERANCE |
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TopAbstractIntroductionFasL In Tissue And...FasL And Autoimmune DiseasesFasL-Transduced Dcs For...Limitations of Fasl UsageSummaryReferences |
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RI and FcRIII). Currently it is accepted that induction of peripheral tolerance by DCs can be achieved by deletion of antigen-specific T cells, induction of T–regulatory cell (Tr1) proliferation that would limit the differentiation of effector autoimmune T cells, and T-cell anergy [155–158]. Other suggested mechanisms used by DCs to induce tolerance are inhibition of T-cell activation by indoleamine 2,3-dioxygenase (IDO) [159,160], upregulation of expression of the inhibitory receptors immunoglobulin-like transcript 3 (ILT3) and ILT4 [161,162], and expression of CD200 (OX-2) [163]. The capacity of DCs to induce tolerance generated considerable interest in the potential use of these cells as therapeutic agents in transplantation, autoimmune disease, and allergy. Different strategies have been developed to reduce allograft rejection and treat different autoimmune diseases using DCs (reviewed in [164–166]). In this section we focus on the strategy of generating DCs that are engineered to over-express FasL, termed "killer" DCs.
FasL has been used to enhance the tolerogenic potential of DCs. T cells upregulate Fas expression upon interaction with DCs and thus become sensitive to Fas/FasL-induced apoptosis. Taking advantage of this phenomenon, it was suggested that FasL-transduced "killer" DCs could serve as a new immunosuppressive agent to deliver a death signal to activated T cells in an antigen-specific manner. As was first shown by Matsue et al. [167], antigen-pulsed DCs transduced with FasL induced apoptosis of the antigen-specific T cells in vitro and suppressed both delayed-type hypersensitivity (DTH) and contact hypersensitivity (CH) responses in an antigen-specific manner in syngeneic mice. When non-pulsed killer DCs were administered to allogeneic mice, only partial inhibition of host immune responses to the allogeneic MHC molecules was achieved. This result led the authors to hypothesize that FasL-transduced donor DCs could eliminate only the host T cells that recognize intact allogeneic MHC molecules via direct presentation by donor APCs and not host T cells that recognize processed alloantigens via the indirect presentation by host APCs. To examine this hypothesis, Matsue et al. created FasL-DC hybrids from donor and recipient DCs (killer DC-DC hybrids) and tested their ability to suppress both alloreactive T-cell populations [168]. Killer DC-DC hybrids inhibited in vitro bidirectional activation of alloreactive T cells between the parental mice strains. When tested in vivo, the killer DC-DC hybrids (A/J x BALB/c) inhibited allo-DTH responses of BALB/c and A/J mice to spleen cells from A/J and BALB/c mice, respectively, and not the allo-DTH responses to spleen cells from another mouse strain (C57BL/6). More important, killer DC-DC hybrids were able to delay the onset of acute GVHD. Min et al. [99] extended these findings by demonstrating the ability of FasL-transduced BALB/c DCs to prolong BALB/c-derived cardiac graft survival in C57BL/6 mice, as was mentioned earlier. In this study, they used mature DCs (MHC IIhigh, C-type lectin-DEC205+, CD40+, CD862+), which compared to immature DCs, express higher Bcl-2 levels. Hoves et al. [169] emphasized the importance of using mature DCs and not immature DCs for FasL transduction, by showing that mature DCs have higher transduction efficiency and reduced sensitivity to Fas/FasL-mediated apoptosis. Mature DCs express Fas, and their protection from apoptosis is probably due to increased expression of anti-apoptotic molecules such as c-FLIP and Bcl-xL [170,171].
Naïve T cells express little cell membrane Fas. The ability of FasL-transduced DCs to eliminate naïve T cells, in an antigen-dependent manner, was tested by Kusuhara et al. [172] using killer DC-DC hybrids (FasL-transduced A/J DCs fused with BALB/c splenic DCs) expressing the I-Ad molecules and naïve CD4+ DO11.10 T cells that recognize the OVA323–339 peptide in an I-Ad–restricted manner. OVA-pulsed killer DC- DC hybrids, but not nonpulsed killer DC-DC hybrids (or OVA-pulsed control DC-DC hybrids with no FasL expression), inhibited activation and induced apoptosis of naïve DO11.10 T cells in vitro. In vivo, syngeneic mice receiving subcutaneous injection of OVA-pulsed killer DC-DC hybrids, but not mice receiving nonpulsed killer DC-DC hybrids or OVA-pulsed control DC DC hybrids, showed reduced viability of the D011.10 T cells that were injected intravenously one day after the injection of killer DC DC hybrids. A study done by Hoves et al. [173] showed that killer DCs could eliminate activated but not resting primary human CD4+ and CD8+ T cells. In this study T cells were activated by anti-CD3 and anti-CD28, not in an antigen-specific manner.
Other APCs (macrophages and B cells) engineered to express FasL have also been shown to be efficient in selective elimination of antigen-specific T cells and induction of tolerance. Zhang et al. [174] demonstrated the ability of allogeneic FasL-expressing macrophages (taken from C57BL/6 lpr/lpr mice to avoid their apoptosis) (H-2b), but not control allogeneic macrophages that do not express FasL, to induce T-cell unresponsiveness in vitro and in vivo of MRL mouse cells (H-2k). T-cell unresponsiveness could not be induced in MRL lpr/lpr mice and was induced only by the alloantigen-specific FasL-expressing macrophages. These findings were confirmed in another experimental system using H-2Db/H-Y T-cell receptor (TCR) transgenic female mice [174]. Kosiewicz et al. [175] showed that B cells pulsed with OVA and engineered to express FasL eliminated OVA-specific T cells (from DO11.10 mice) that were injected into BALB/c mice and suppressed DTH response in an antigen-specific manner.
As was observed by Georgantas et al. [176], induction of peripheral T-cell tolerance to a specific antigen can also be achieved by intramuscular injection of gelatin nanoparticles that contain two plasmid DNA vectors, one encoding FasL and the other encoding the specific antigen. These nanoparticles (which also contained transferrin cell ligand, calcium, and chloroquine) mediated the transfection of the FasL DNA and the model antigen ß-galactosidase (ß-gal) DNA to APCs in the draining lymph nodes. Mice injected with DNA-gelatin nanoparticles containing ß-gal DNA, alone, developed a strong ß-gal-specific CTL response that was not seen when FasL DNA was co-delivered with ß-gal DNA. The induction of T-cell tolerance is antigen-specific, as was shown by the inability of the DNA-gelatin nanoparticles containing both FasL and ß-gal DNA to block immunity to a second antigen injected at a separate site.
Not all the attempts to use FasL-expressing allogeneic DCs to specifically delete alloreactive T cells and confer tolerance have met with success. As was shown by Buonocore et al. [177,178], subcutaneous injection of immature FasL-transduced DCs from C57BL/6 lpr mice to bm12 or bm13 mice induced allogeneic T-cell hyperactivation and acute rejection of C57BL/6 skin allografts, respectively.
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In the case of liver transplantation, although the liver is highly susceptible to FasL, Li et al. [105] showed that low levels of FasL expression on allogeneic livers (~10%) are not toxic and that significant prolongation in their survival can be achieved. In contrast, when higher levels of FasL (>10%) are expressed on the allogeneic livers, lethal hepatitis developed.
Using FasL-"armed" cells to specifically delete alloreactive T cells might also nonspecifically eliminate other T cells or other cells expressing Fas receptor, especially immune cells. As was shown by Cheng et al. [187], transgenic mice constitutively expressing FasL in mature T cells developed normally and were healthy, and although they showed reduced T-cell numbers, they contained mature functional T cells that were resistant to Fas/FasL-mediated apoptosis. DCs, B cells, and macrophages can also resist Fas/FasL-mediated apoptosis by upregulating the expression of anti-apoptotic molecules such as c-FLIP and Bcl-xL [170, 171, 188–190]. Also, as was mentioned above, transduction of limited numbers of cells with FasL or locally controlled FasL expression might reduce its side effects.
Aside from its ability to induce apoptosis, FasL has also pro-inflammatory characteristics and can induce the cellular release of multiple pro-inflammatory chemokines and cytokines [191–193]. As was demonstrated in vivo, FasL indirectly chemo-attracts neutrophils by inducing apoptosis of Fas-expressing cells in its microenvironment [194]. Apoptotic cells then release chemotactic factors and attract neutrophils. As was observed in several studies, overexpression of FasL on allogeneic cells and organs led to their accelerated rejection [97, 109, 110, 115, 116, 177, 178]. The ability to control and regulate the opposing pro- and anti-inflammatory effects of FasL will enable enhancing FasL’s anti-inflammatory activity, block its pro-inflammatory activity, and prevent allograft rejection. Studies on immune-privileged sites have shown that constitutive expression of anti-inflammatory cytokines such as TGF-ß, alpha-melanocyte-stimulating hormone (MSH) and vasoactive intestinal peptide (VIP) contributes to and complements the function of FasL [195–197]. As was demonstrated by Chen et al. [198], the pro-inflammatory activity of FasL can be regulated by TGF-ß, and in order to protect colon carcinoma cells from immune destruction, the expression of both FasL and TGF-ßis needed. Therefore, one strategy to enhance the anti-inflammatory activity of FasL is to mimic its supportive microenvironment at immune-privileged sites.
As was shown on human endothelial cells, Fas/FasL-induced release of the chemokines IL-8 and monocyte chemo-attractant protein-1 (MCP-1) is triggered via a caspase-independent pathway [199]. Other studies done in order to clarify the difference between the Fas/FasL signaling pathway leading to apoptosis and the Fas/FasL signaling pathway leading to the release of pro-inflammatory cytokines will enable the design of specific inhibitors to the Fas/FasL inflammatory pathway.
Several studies have shown that the inflammatory activity of FasL is mediated by the membrane-bound but not the soluble form of FasL [186, 194, 200, 201]. Hence, it is possible that local administration of sFasL will downregulate the pro-inflammatory activity of mFasL.
The transplantation site might also be important. As in immune-privileged sites, the microenvironment into which the cells or grafts are transplanted might support the protective role of FasL or antagonize it. As was observed by Seino et al. [202], FasL-transduced cells were rejected when they were transplanted subcutaneously into syngeneic mice but survived when they were transplanted under the kidney capsule. This consideration should be taken into account in transplantation of FasL-transduced DCs.
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) clearly demonstrate the ability of FasL to protect allogeneic grafts from immune rejection, either by expressing FasL on the allogeneic tissue itself or by administration of FasL-transduced allogeneic donor DCs. They also give a clear indication of the potential for FasL to be used in treatment of several autoimmune diseases. However, as indicated by certain other studies, ectopic expression of FasL triggers an inflammatory response that leads to the destruction of the FasL-expressing tissue. The inability to control the outcome of FasL expression temporarily limits the use of this strategy, until it becomes possible to separate the anti-inflammatory beneficial functions of FasL from its detrimental pro-inflammatory functions. A balance between the anti-inflammatory and inflammatory responses at the site of transplantation probably determines the outcome of FasL expression, and strategies aiming to change this balance toward the anti-inflammatory effect of FasL should be developed. One of these strategies might be to mimic the anti-inflammatory microenvironment of FasL in immune-privileged sites, as was done by Chen et al. [198] (mentioned earlier). Chen et al. showed that colon carcinoma cells could be protected from immune destruction by co-expressing FasL with the anti-inflammatory cytokine TGF-ß known to be expressed in immune-privileged sites. The co-expression of FasL with other known anti-inflammatory and immunosuppressive cytokines in immune-privileged sites should be tested as well. Also, FasL is constitutively expressed in immune-privileged sites, and as was shown in the studies presented in this review, FasL gene transfer using adenoviruses achieved only transient expression of FasL. To be able to confer immune privilege on allogeneic grafts, it is possible that permanent or extended FasL expression will be needed. Furthermore, just as immune-privileged sites and tumor cells are equipped with several protective mechanisms, in order to block its rejection, allogeneic tissue should also be equipped with several immuno-suppressive mechanisms in addition to FasL expression.
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Aside from the pro-inflammatory function of FasL that was discussed earlier, inflammation is part of the recovery process from the surgery itself. To avoid immediate rejection due to the inflammatory response, a short treatment with anti-inflammatory drugs immediately after or together with the transplantation of the FasL-expressing allograft is suggested.
In conclusion, the concept of using FasL as immunosuppressive molecule is still in its infancy. Gaining a better understanding of the various functions of FasL may enable the safe use of FasL for transplantation and treatment of inflammatory and autoimmune diseases.
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