T. Deguchi

Rapamycin Causes Upregulation of Autophagy and Impairs Islets Function Both In Vitro and In Vivo

Autophagy is a lysosomal degradation process of redundant or faulty cell components in normal cells. However, certain diseases are associated with dysfunctional autophagy. Rapamycin, a major immunosuppressant used in islet transplantation, is an inhibitor of mammalian target of rapamycin and is known to cause induction of autophagy. The objective of this study was to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β cells of green fluorescent protein–microtubule‐associated protein 1 light chain 3 transgenic mice. Rapamycin reduced the viability of isolated β cells and down‐regulated their insulin function, both in vitro and in vivo. In addition, rapamycin increased the percentages of apoptotic β cells and dead cells in both isolated and in vivo intact islets. Treatment with 3‐methyladenine, an inhibitor of autophagy, abrogated the effects of rapamycin and restored β‐cell function in both in vitro experiments and animal experiments. We conclude that rapamycin‐induced islet dysfunction is mediated through upregulation of autophagy, with associated downregulation of insulin production and apoptosis of β cells. The results also showed that the use of an autophagy inhibitor abrogated these effects and promoted islet function and survival. The study findings suggest that targeting the autophagy pathway could be beneficial in promoting islet graft survival after transplantation.

Rapamycin induces autophagy in islets: relevance in islet transplantation.

Islet transplantation can provide insulin independence in patients with type 1 diabetes mellitus. However, islet allograft recipients exhibit a gradual decline in insulin independence, and only 10% do not require insulin at 5 years. This decline may reflect drug toxicity to islet beta cells. Rapamycin, a central immunosuppressant in islet transplantation, is a mammalian target of rampamycin inhibitor that induces autophagy. The relative contributions of autophagy in transplanted islets are poorly understood. Therefore, in the present study we sought to evaluate the effects of rapamycin on islet beta cells. Rapamycin treatment of islets resulted in accumulation of membrane-bound light chain 3 (LC3-II) protein, an early marker of autophagy. In addition, rapamycin treatment of isolated islets elicited not only reduction of viability but also downregulation of in vitro potency. To further examine the occurrence of autophagy in rapamycin-treated islets, we used GFP (green fluorescent protein)-LC3 transgenic mice that express a fluorescent autophagosome marker. The GFP-LC3 signals were markedly increased in rapamycin treated islets compared with control islets. In addition, to show improvement by blockade of autophagic signaling, islets were treated with rapamycin in the presence of 3-methyladenine, which inhibits autophagy. Thereafter, both islet viability and islet potency were dramatically improved. The number of GFP-LC3 dots clearly increased after 3-MA treatment. Thus, rapamycin treatment of islets induces autophagy in vitro. This phenomenon may contribute to the progressive graft dysfunction of transplanted islets. Therapeutically targeting this novel signaling may yield significant benefits for long-term islet survival.

In Vitro and in Vivo Prevention of Human CD8+ CTL‐Mediated Xenocytotoxicity by Pig c‐FLIP Expression in Porcine Endothelial Cells

Overcoming cell‐mediated immunity, especially of human CD8+ CTLs, is important for the success of xenotransplantation. Our group has previously reported that the cytotoxicity of human CD8+ CTLs against pig endothelial cells (PEC) is highly detrimental and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c‐FLIP) was originally identified as an inhibitor of death‐receptor signaling through binding competition with caspase‐8 for recruitment to Fas‐associated via death domain (FADD). Two major c‐FLIP variants result from alternative mRNA splicing: a short, 26‐KDa protein (c‐FLIPS) and a long, 55‐KDa form (c‐FLIPL). The cytoprotective effects of c‐FLIPS/L in xenograft cells remain controversial. This study demonstrates that the overexpression of c‐FLIPS/L genes markedly suppress human CD8+ CTL‐mediated xenocytotoxicity and, in addition, the cytoprotective effects of c‐FLIPL appear to be significantly stronger than those of c‐FLIPS. Furthermore, to prove the prolonged effects of xenograft survival, PEC transfectants with c‐FLIPS/L genes were transplanted under rat kidney capsules. Prolonged survival was elicited from FLIPS/L transfectants, whereas parental PEC was completely rejected through day 5, posttransplant. Thus, intracellular remodeling with the overexpression of c‐FLIPS/L in xenograft cells may avoid innate cellular attacks against xenografts and facilitate long‐term xenograft survival.

Prolonged survival of pig islets xenograft by adenovirus-mediated expression of either the membrane-bound human FasL or the human decoy Fas antigen gene.

Abstract:  Background:  Pig islets are considered an attractive alternative treatment for patients with Type 1 diabetes. However, pig islet xenografts, transplanted into non‐human primates, are directly rejected by cell‐mediated processes. We have previously reported that cell‐mediated xenograft‐rejections, and especially human CD8+ cytotoxic T lymphocytes (CTL)‐mediated cytotoxicity, are highly detrimental to pig xenograft cells. Moreover, we have explored novel strategies for the prevention of CTL killing by overexpression of either human decoy Fas antigen or membrane‐bound human FasL in pig endothelial cells. In this study, we assessed the cytoprotective effects of these molecules for pig islets both in vitro and in vivo.

Adenoviral-Mediated Overexpression of Either Membrane-Bound Human FasL or Human Decoy Fas Can Prolong Pig Islet Xenograft Survival in a Rat Transplant Model

The success of pancreatic islet transplantation is limited because of the severe shortage of allogeneic pancreas donors. Accordingly, pig islets are considered to be an attractive, promising alternative. However, cell-mediated immunity, especially CD8+ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a formidable barrier to prevent long-term islet survival in xenograft recipients. Therefore, it is particularly important to explore methods to specifically prevent cell-mediated immunity against pig islets. Our group previously demonstrated that the overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig endothelial cells prevented CTL xenocytotoxicity. In this study, we assessed the cytoprotective effects of adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig islets to inhibit CTL xenocytotoxicity. The CTL-mediated killing of pig islets infected with an adenoviral vector carrying either membrane-bound human FasL or human decoy Fas was significantly reduced compares with that of control pig islets transfected with adenoviral vector encoding enhanced green fluorescent protein (EGFP). Moreover, we transfected pig islets with these molecules to confirm their cytoprotective effects in in vivo studies. The significant long-term survival of pig islets expressing these molecules was elicited through days 3 to 5 posttransplantation. Thus, these results demonstrated that the remodeling of either death receptor or death ligand on pig islets by adenoviral gene transfer prevented innate cellular immunity against xeno-islet grafts facilitating long-term xenograft survival.

Pig Cellular FLICE-like Inhibitory Protein (c-FLIP) Overexpression in Pig Xenograft Cells Induces Resistance to Human CD8+ Cytotoxic T Lymphocyte-Mediated Xenocytotoxicity

Although the use of organs from alpha1,3-galactosyltransferase gene knockout pigs may prolong xenograft survival, resulting in overcoming antibody-mediated hyperacute rejection, pig xenografts will be destroyed directly by cell-mediated immunity, such as NK cells, macrophages, and CD8+ cytotoxic T lymphocytes (CTLs). Therefore, conquering cell-mediated immunity, especially of human CD8+ CTLs, is of particular importance to the success of long-term xenograft survival. We have previously reported that the cytotoxicity of human CD8+ CTLs is strong against pig endothelial cells (PEC) and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as a potent inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP S) and a long, 55-kDa form (c-FLIP L). The present study demonstrated that overexpression of c-FLIP S/L genes in PEC markedly suppressed human CD8+ CTL-mediated xenocytotoxicity; moreover, the cytoprotective effects of c-FLIP L appeared to be significantly stronger than those of c-FLIP S. Furthermore, to prove the in vivo prolongation effects of xenograft survival, we transplanted PEC transfectants with c-FLIP(S/L) genes under the rat kidney capsule. Prolonged survival was displayed by xenografts of FLIP S/L PEC transfectants, whereas xenografts of parental PEC were completely rejected by day 5 posttransplantation. Thus, intracellular blocking of death receptor-mediated apoptotic signals by overexpression of c-FLIP S/L in xenograft cells may prevent innate cellular attacks against xenografts opening the window of opportunity for long-term xenograft survival.

In Vivo Controlling of Cellular Response to Pig Islet Xenografts by Adenovirus-Mediated Expression of Either Membrane-Bound Human FasL or Human Decoy Fas

The critical problem with clinical islet transplantation for patients with type 1 diabetes is the severe shortage of human donors. Pig islet xenotransplantation has the potential to provide a virtually unlimited source of donor pancreata. However, our previous studies demonstrated that cell-mediated rejection, especially human CD8(+) cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a major obstacle for long-term islet xenograft survival. Moreover, we have demonstrated that the overexpression of either membrane-bound human FasL (mFasL) or human decoy Fas antigen (decoy Fas) in pig islets not only prevented CTL xenocytotoxicity in vitro, but also prolonged histological survival of pig islet xenografts in vivo. Therefore, the aim of the present study was to determine whether adenoviral transfer of these genes into pig islets ex vivo prior to transplantation had a beneficial effect on posttransplantation glycemic control of diabetic recipients. Isolated pig islets were transfected with adenovirus vector carrying complementary DNA (cDNA) of either mFasL or decoy Fas. The transfected islets were transplanted under the kidney capsule of diabetic recipient rats. Rats transplanted with either mFasL- or decoy Fas-transfected pig islet grafts showed significantly suppressed blood glucose levels from 12 hours to 18 hours posttransplantation compared with control groups transplanted with empty vector-transfected pig islets. Unfortunately, blood glucose levels of these groups were increased, with no significant difference observed at 24 hours posttransplantation. However, transgenic expression of these molecules with clinically tolerable amount of immunosuppressants may be more effective to achieve islet xenograft survival in the future.

Abstract 4747: MUC1, remodeled to express α-gal epitopes immune-based therapy, can elicit both significant prolonged survival and effective antibody production against multiple tumor-associated antigens

MUC1, a membrane bound mucin glycoprotein, is overexpressed and aberrantly glycosylated in more than 80% of human pancreatic carcinoma, has generated considerable interest as a potential target for immunotherapy. MUC1 immune-based therapies have not been successful, because immunity toward tumor-associated antigens (TAAs) in cancer patients is weak and the presentation of TAAs to the immune system is poor. Human natural antibody, Anti-Gal is an IgG known to be present in large amounts in normal subjects and patients with malignancies, comprising ∼1% of serum circulating IgG. Anti-Gal specifically interacts with α-gal epitopes (Galα1, 3Galβ1, 4GlcNAc-R), synthesized by α1, 3 galactosyltransferase (α1,3GT) on cell surface glycolipids and glycoproteins. MUC1 remodeled to express α-gal epitopes (α-gal MUC1), because MUC1 has five potential sites of N-glycans, can bind anti-Gal in situ at the vaccination site. Such interaction would enhance the recognition by APCs, resulting in more effective vaccination. We previously investigated the effectiveness of elicitation of both antibody production and T cell responses against MUC1 antigen by α-gal MUC1 vaccination. Present study addresses the usefulness of α-gal MUC1 vaccination for prolongation of survival and antibodies production, including anti-MUC1 and Abs toward other TAAs. A human pancreatic cancer cell line, PANC1, which expresses MUC1 was employed and transfected with α1,3GT gene (α-gal PANC1). High anti-Gal α1,3GT KO mice, which displayed anti-Gal titers similar to those found in humans were generated by immunization of pig tissue. These mice were vaccinated with either 1 × 10 6 irradiated parental PANC1 (control group) or α-gal PANC1 (α-gal group). To demonstrate in vivo tumor destruction by α-gal PANC1 vaccination, vaccinated mice were injected s.c. with 0.5 × 10 6 B16 melanoma cells, which were transfected with MUC1 gene. The mean survival of either control or α-gal group were found to be 21.1±10.5 days or 41.1±10.4 days, respectively, and the survival of α-gal group was significantly prolonged (control vs. α-gal group; p=0.003). To understand the induced antibody response for other TAAs, immunostained PANC1 proteins were investigated in Western blots with mice serum before/after vaccination. Although the serum from control group fail to display the significant bands, which was reflected by both anti-MUC1 Ab and anti-unknown TAAs Abs, the serum from α-gal group contained multiple antibodies that bound to not only MUC1 but also different unknown TAAs. We conclude that vaccination with tumor lysate remodeled to express α-gal epitopes can effectively upregulated immunogenicity of multiple TAAs, including MUC1 but also other unknown TAAs and may provide a chance to develop the potential immunotherapy for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4747.

Intracellular and Extracellular Remodeling Effectively Prevents Human CD8+Cytotoxic T Lymphocyte-Mediated Xenocytotoxicity by Coexpression of Membrane-Bound Human FasL and Pig c-FLIPL in Pig Endothelial Cells

Human CD8(+) cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, which participates in xenograft rejection, is mediated mainly by the Fas/FasL apoptotic pathway. We previously developed methods to inhibit human CTL xenocytotoxicity by extracellular remodeling using overexpression of membrane-bound human FasL on pig xenograft cells, and by intracellular blockade of death receptor-mediated apoptotic signals, such as the Fas/FasL pathway using the pig c-FLIP(L) molecule. To investigate the cooperative effects of both membrane-bound FasL and pig c-FLIP(L), we cotransfected both genes into pig endothelial cells (PEC). The double remodeling with these molecules effectively prevented CD8(+) CTL killing. Although double transfectants and single high transfectants of either membrane-bound FasL or c-FLIP(L) gene displayed similar inhibition of CTL cytotoxicity, the expression levels of these 2 molecules in double transfectants were almost half the expression levels of single transfectants. Furthermore, to show in vivo prolongation of xenograft survival, we transplanted PEC transfectants under the rat kidney capsule. Prolonged survival was displayed by PEC double transfectant xenografts whereas those from either parental PEC or MOCK (vehicle control) were completely rejected by day 5 posttransplantation. These data suggested that intracellular and extracellular remodeling by coexpression of membrane-bound FasL and pig c-FLIP(L) in xenograft cells may prevent an innate cellular response to xenografts. The gene compatibility of these molecules to generate transgenic pigs may be sufficient to create a window of opportunity to facilitate long-term xenograft survival.

Adenovirus-Mediated Gene Expression of the Human c-FLIPL Gene Protects Pig Islets Against Human CD8+ Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP(S/L) genes, which are potent inhibitors of death receptor-mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP(L) seem to be significantly stronger than those of c-FLIP(S). Accordingly, in the present study, expression of c-FLIP(L) was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% +/- 15.9% and 64.0% +/- 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP(L) gene were markedly protected from lysis (30.5% +/- 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP(L) with adenovirus. Pig islets expressing c-FLIP(L) are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.