Pancreatic Tissue Transplanted in TheraCyte ™ Encapsulation Devices Is Protected and Prevents Hyperglycemia in a Mouse Model of Immune-Mediated Diabetes

Abstract

Type 1 diabetes (T1D) is characterized by destruction of glucose-responsive insulin-producing pancreatic β-cells and exhibits immune infiltration of pancreatic islets, where CD8 lymphocytes are most prominent. Curative transplantation of pancreatic islets is seriously hampered by the persistence of autoreactive immune cells that require high doses of immunosuppressive drugs. An elegant approach to confer graft protection while obviating the need for immunosuppression is the use of encapsulation devices that allow for the transfer of oxygen and nutrients, yet prevent immune cells from making direct contact with the islet grafts. Here we demonstrate that macroencapsulation devices (TheraCyte^™) loaded with neonatal pancreatic tissue and transplanted into RIP-LCMV.GP mice prevented disease onset in a model of virus-induced diabetes mellitus. Histological analyses revealed that insulin-producing cells survived within the device in animal models of diabetes. Our results demonstrate that these encapsulation devices can protect from an immune-mediated attack and can contain a sufficient amount of insulin-producing cells to prevent overt hyperglycemia.

Introduction

Islet transplantation represents an important approach to reinstating glucose-responsive insulin production in diabetic patients, and its recently increasing success rates are encouraging (1). If all other factors are optimal (donor management, the functional state of the donor organ, the surgical procedure, and intraoperative management of the recipient), the major reason for transplant failure is rejection (5). The transplanted cells are a continuous source of HLA alloantigens capable of inducing a rejection response. The allograft continuously activates the immune system, causing lifelong overproduction of cytokines, constant cytotoxic activity, and sustained alteration in the graft vasculature. The persistence of islet-reactive autoimmune cells (6) in diabetic patients also constitutes a major caveat for the success of islet transplantation. In order to ensure graft survival, strong immunosuppressants are needed, some of which exhibit β-cell toxicity (11) and expose the patients to increased risk of cancer (9) and infectious diseases. A proposed solution is to establish a physical barrier between graft and immune cells, ideally through a membrane permeable to nutrients, oxygen, and peptides, but impermeable to immune cells and, thus, markedly reduce the need for pharmacological immunosuppression.

Here we used semipermeable macroencapsulation devices (TheraCyte^™ as well as similar, newer generation devices) to transplant neonatal pancreatic tissue from either wild-type C57BL/6 or RIP-LCMV.GP donor mice into RIP-LCMV.GP recipients and subsequently challenged the encapsulated grafts with a virus-induced CD8 T-cell-mediated immune attack as a model of T1D. In the RIP-LCMV.GP mouse strain, infection with lymphocytic choriomeningitis virus (LCMV) induces a cytotoxic T-lymphocyte (CTL) response against the viral glycoprotein (GP) that is transgenically expressed on the endogenous β-cells (14). Consequently, RIP-GP mice develop hyperglycemia 7–8 days postinfection with LCMV (14).

We chose to transplant neonatal pancreatic tissue, as its resistance to hypoxia is higher than adult tissue (4), resembling that of progenitor cells (10). This approach helps to overcome the limited oxygen supply in the immediate posttransplantation period. In all groups, TheraCyte^™ devices offered good engraftment and long-term survival of the transplanted endocrine tissue, conferring effective protection against immune attack, as revealed by histological analyses. Furthermore, graft-derived insulin lowered blood glucose and prevented diabetes onset in some of the recipients. Upon surgical explantation of the grafts, these mice developed overt hyperglycemia.

Hence, we were able to show for the first time that pancreatic tissue encapsulated in an immunoprotective device can maintain euglycemia in mice that are rendered diabetic in a virus-induced model of immune-mediated diabetes.

Materials and Methods

Mice

RIP-LCMV.GP (14) and C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were bred and housed under specific pathogen-free conditions at the La Jolla Institute for Allergy and Immunology. Both male and female mice were used as donors and recipients. The sex of the recipient mice in the individual experiments was as follows: 3 males/2 females (Fig. 1A); 4/3 (Fig. 1B); 3/2 (Fig. 1C); all males (Fig. 1D E). Both RIP-LCMV.GP and C57BL/6 mice share the same background (C57BL/6) and are syngeneic to each other. All experiments were approved by the Institutional Animal Care and Use Committee at the La Jolla Institute for Allergy and Immunology (MvH AP150).

Figure 1.

Pancreatic tissue transplanted in PTFE encapsulation devices (TheraCyte^™) protect recipient mice from hyperglycemia. All experiments were performed with RIP-LCMV.GP mice. In order to induce diabetes through immune-mediated destruction of endogenous islets, mice were infected with LCMV Armstrong 10⁴ PFU IP. Infection was performed 20 or 21 days after implantation of immunoprotective devices. Curves represent blood glucose values following LCMV infection. (A) Black symbols: control mice, no devices; (B) red symbols: devices containing C57B1/6 grafts (syngeneic, no immune attack); (C) green symbols: devices containing RIP-LCMV.GP grafts (syngeneic, autoimmune attack); (D) control mice without implanted devices (black) and mice with devices containing RIP-LCMV.GP grafts (green). The devices were explanted 30 days after surgery (10 days after infection); (E) control mice without implanted devices (black), mice with intact devices containing RIP-LCMV.GP grafts (green), and mice with perforated devices containing RIP-LCMV.GP grafts (blue). (A–D) TheraCyte^™ devices, (E) similar, newer generation devices.

Graft Preparation

Newborn (< 72 h) wild-type C57BL/6 or RIP-LCMV. GP mice were sacrificed in strict accordance with the PHS Policy on Humane Care and Use of Laboratory Animals of the National Institutes of Health. The pancreata were carefully removed and freed of adjacent fat tissue under a dissection microscope. Six to eight organs were pooled, and pancreata were minced using a McIlwain^™ Tissue Chopper (Ted Pella, Inc., Redding, CA, USA). Minced pieces were washed and cultured in a cell incubator at 37°C and 5% CO₂ in 30-mm Petri dishes (BD, Franklin Lakes, NJ, USA) with serum-free RPMI supplemented with 5 IU/ml DNAse A (Roche, Kaiseraugst, Switzerland), penicillin, and streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Following overnight culture, the pancreatic tissue was resuspended and filled into a 1-ml plastic syringe (BD) and carefully loaded into a prewetted 20-μl polytetrafluoroethylene (PTFE) encapsulation device (or similar, newer generation device; TheraCyte^™; Laguna Hills, CA, USA) using a 20-gauge venous catheter. Loaded devices were sealed with biological-grade silicone adhesive (NuSil®, Carpinteria, CA, USA) and deposited in serum-free RPMI until implantation. Some of the devices were perforated using an 18-gauge cannula, to allow for immune cell infiltration. All operations were performed under a laminar flow hood or biologic safety cabinet.

Implantation of the Devices

Overnight fasted mice were anesthetized using a mix of 100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine (both from Bayer, Leverkusen, Germany) administered by intraperitoneal (IP) injection. The device was implanted in a dorsal subcutaneous pocket created by blunt dissection. Wounds were closed with wound clips (Kent Scientific, Torrington, CT, USA) and sealed with wound adhesive (Johnson & Johnson, New Brunswick, NJ, USA). Control animals either received no graft-bearing encapsulation device or were transplanted with loaded devices that had been perforated. Mice were kept warm under heat lamps until full recovery.

LCMV Infection

Three weeks after implantation of the devices, mice were injected (IP) with 10⁴ PFU of LCMV strain Armstrong (14).

Blood Glucose Monitoring

Nonfasting blood glucose values were measured twice weekly by means of a commercially available glucometer (Accu-Chek^™; Roche, Kaiseraugst, Switzerland) using blood derived from the tail vein.

Explantation of the Devices

In some mice, devices were explanted to demonstrate that lower blood glucose values were maintained by the transplanted cells. Pre- and postoperative steps were identical to the implantation procedure. The device was carefully freed from surrounding skin and fascia by sharp dissection, and electrocautery was used for hemostasis. Devices were removed from the mouse and immediately placed in neutral-buffered formalin (Sigma-Aldrich).

Sacrifice of Mice and Harvesting of Encapsulation Devices and Tissue

Mice were sacrificed via CO₂ overdose and decapitated, in compliance with the PHS Policy on Humane Care and Use of Laboratory Animals of the National Institutes of Health. Encapsulation devices were removed by carefully releasing them from the surrounding tissue using surgical scissors. In general, mice showing signs of overt diabetes were sacrificed, and devices were explanted, where applicable.

Histological Analyses

Explanted devices were fixed with neutral-buffered formalin, processed with a Leica Tissue Processor (Leica ASP300S; Leica, Wetzlar, Germany), and embedded in paraffin blocks. Paraffin block samples were sectioned at 5-μm thickness. H&E staining was performed using a Leica autostainer (ST5020; Leica) and the Leica Infinity Stain Kit. Immunohistochemical (IHC) staining for insulin (A0564; DAKO, Glostrup, Denmark) was performed using a fully automated Leica BOND III stainer with 3,3′-diaminobenzidine (DAB) as the chromogen. The lymphocyte marker CD8 was stained with a commercially available monoclonal antibody (1:100; MA1-70041; Pierce Antibodies, Rockford, IL, USA), and detection was done using a goat anti-rat IgG secondary antibody (1:1,000; ab150157; Abcam, Cambridge, MA, USA) coupled to FITC.

IHC images were captured by a digital slide scanner Nanozoomer (Hamamatsu, Shizuoka, Japan). Immunofluorescent (IF) images were taken using a fluorescence microscope (Nikon, Tokyo, Japan) and a computer-based microscopy software (Nikon ExAct^™; Tokyo, Japan).

Results

Pancreatic Tissue Inside a PTFE Encapsulation Device Is Able to Maintain Systemic Euglycemia

To test the engraftment of encapsulated neonatal pancreatic tissue in the RIP-LCMV.GP mouse model, we initially transplanted TheraCyte^™ devices loaded with neonatal pancreatic tissue from newborn syngeneic C57BL/6 wild-type mice into 6- to 8-week-old RIP-LCMV.GP recipients. Three weeks after the implant surgery, transplanted RIP-GP mice were infected with LCMV, and blood glucose values (BGV) were measured to evaluate development of hyperglycemia. Notably, while all control mice developed hyperglycemia, mice that had been implanted with a device loaded with neonatal pancreatic tissue remained largely euglycemic (Fig. 1A, B), suggesting that the pancreatic tissue within the implanted device was able to maintain euglycemia despite destruction of the endogenous β-cells.

PTFE Encapsulation Devices Protect Endocrine Tissue From Immune-Mediated Destruction

In order to assess whether these devices are able to protect transplanted β-cells from islet-specific immunity, we loaded neonatal pancreatic tissue from RIP-LCMV. GP mice into TheraCyte^™ devices and transplanted the devices into RIP-LCMV.GP mice. Following LCMV infection, BGV was determined, and diabetes development was monitored. Notably, mice that were engrafted with tissue from RIP-LCMV.GP donors loaded into PTFE devices had lower BGV, with some of them maintaining euglycemia as long as 95 days posttransplantation (74 days post-LMCV infection) (Fig. 1C). In comparison, all control animals receiving no graft (Fig. 1A) or intentionally perforated devices loaded with neonatal pancreatic tissue (Fig. 1E) developed diabetes rapidly, demonstrating a potent destruction of endogenous pancreatic tissue by the anti-LCMV immune response in RIP-LCMV.GP mice. In some of the mice we removed the graft-bearing device in a second surgical procedure, and in most cases, BGV immediately increased upon explantation of the devices (Fig. 1D, E). These observations suggest that encapsulation devices effectively protect against β-cell-specific immune attacks.

Histological Evidence of Viable Cells and Insulin Production Within PTFE Encapsulation Devices

To further analyze whether insulin-producing cells survive within the encapsulation devices, mice were sacrificed, and explanted devices were embedded in paraffin and sectioned. Sections were stained with H&E in order to analyze the morphology of the cells inside the devices. Live cell clusters could be identified within the devices containing C57BL/6-derived pancreatic tissue (Fig. 2A) as well as the devices containing RIP-LCMV.GP-derived pancreatic tissue (Fig. 2B). Importantly, these clusters could be identified until 208 days posttransplantation (Fig. 2C). Following antigen retrieval, we performed immunohistochemical analysis of insulin production. Notably, we found insulin-producing cell clusters within the devices containing C57BL/6-derived as well as RIP-LCMV.GP-derived pancreatic tissue (Fig. 2A, B), suggesting that the anti-LCMV immune response was unable to target insulin-producing cells inside the device. Collectively, histological analyses reveal that insulin-producing cell clusters can survive within intact encapsulation devices.

Figure 2.

Pancreatic tissue transplanted in PTFE encapsulation devices contain insulin-producing cell clusters. Representative histologies of explanted TheraCyte^™ devices are displayed. Pancreatic tissues from nontransgenic C57Bl/6 as well as RIP-LCMV.GP mice survive inside the encapsulation devices and form insulin-producing cell clusters. Devices were removed at 95 days (A and B) and 208 days (C) posttransplantation, respectively. (A and B) H&E stainings are shown in the upper right and at the bottom (higher magnification). Immunohistochemical analysis of insulin forming cell clusters is displayed in the upper left. (D and E) Slides A and B were immunostained for CD8. (D) In C57/Bl6 grafts, no CD8⁺ cells are observed in the vicinity of the encapsulated grafts (red dotted circles), whereas (E) in RIP-LCMV.GP grafts, CD8⁺ cells (red arrows) group in the immediate vicinity of the grafts, on the exterior aspect of the encapsulation device.

CD8⁺ T Cells Migrate to the Pericapsular Area around the Devices

Slides were stained for the presence of CD8⁺ T cells in proximity to the graft. We observed many CD8⁺ T cells grouped in the immediate vicinity of the devices containing RIP-LCMV.GP tissue, on the exterior side of the encapsulation device (Fig. 2E). In contrast, no CD8⁺ T cells were present in the vicinity of encapsulated C57BL/6-derived tissue (Fig. 2D). These results suggest that antigens are able to escape the encapsulation device and trigger an immune response to the graft. However, these cells do not penetrate the encapsulation membrane, and the graft appears to be protected.

Discussion

Here we report the use of PTFE immune isolation devices in an experimental setting to test for their immunoprotective capacity when facing β-cell-specific immune attack. We have employed the RIP-LCMV.GP mouse in a virus-induced diabetes model (14) that allows for an elegant recapitulation of the immune attack seen in autoimmune diabetes.

We transplanted encapsulated neonatal pancreatic tissue from syngeneic C57BL/6 or from transgenic RIP-LCMV.GP donor mice into RIP-LCMV.GP recipients to assess engraftment in a nonimmune setting and to assess protection from a β-cell-specific immune attack that resembles the recurrent autoimmunity in human clinical islet transplantation. In all groups, the encapsulation devices offered good engraftment and long-term survival of the transplanted endocrine tissue, while also conferring protection against T-cell-mediated immune attack. To our knowledge, we are the first group to combine the concept of immune isolation using semipermeable membranes with a virus-induced model of diabetes in order to show that pancreatic, endocrine tissues transplanted in PTFE encapsulation devices are able to confer glycemic control despite the presence of β-cell-specific immunity.

In some of the explanted devices, we observed an accumulation of CD8⁺ T cells surrounding the membrane. This suggests that there is potentially leakage of peptides derived from the graft sufficient to attract host CD8⁺ T cells. Previous work has shown that such a leakage can lead to an immune attack (7), yet it is still unknown what antigens might be passing through the membrane and how exactly they might activate host immune cells.

Different methods for encapsulation have been evaluated in several animal models (3). In one macroencapsulation study, islet allografts were transplanted into the epididymal fat pad of streptozotocin-induced diabetic mice. Four weeks posttransplant, mice attained normoglycemia lasting up to 12 weeks (12). In a nonhuman primate study, porcine islet microcapsules were placed into the intraperitoneal cavity of 16 diabetic cynomolgus primates, and exogenous insulin requirement decreased to 36% at 12 weeks and 43% at 23 weeks compared with controls (8). The TheraCyte^™ device has previously been shown by various groups to allow engraftment and immune protection in different experimental settings (2,10,13). The TheraCyte^™ device was able to sustain islets, protect allogeneic cells from immune attack, and provide treatment for diabetic-mediated weight loss in both BioBreeding rats and streptozotocin-induced diabetic rats (13). Here we have further demonstrated that these devices, as well as their newer generation counterparts, protect highly complex tissues such as neonatal endocrine tissue in the context of an immune attack that closely resembles the T-cell-mediated autoimmune attack found in human T1D. Further development of these and similar macroencapsulation devices may ultimately provide the means for widespread clinical application of a cellular therapy for T1D.