Intrapleural Transplantation of Allogeneic Pancreatic Islets Achieves Glycemic Control in a Diabetic Non-Human Primate

Abstract

Clinical islet transplantation has relied almost exclusively on intraportal administration of pancreatic islets, as it has been the only consistent approach to achieve robust graft function in human recipients. However, this approach suffers from significant loss of islet mass from a potent immediate blood mediated inflammatory response (IBMIR) and a hypoxic environment. To avoid these negative aspects of the portal site, we explored an alternative approach in which allogeneic islets were transplanted into the intrapleural space of an NHP, treated with an immunosuppression regimen previously reported to secure routine survival and tolerance to allogeneic islets in NHP. Robust glycemic control and graft survival were achieved for the planned study period of >90 days. Our observations suggest the intrapleural space provides an attractive locale for islet transplantation due to its higher oxygen tension, ability to accommodate large transplant tissue volumes, and a lack of IBMIR-mediated islet damage. Our preliminary results reveal the promise of the intrapleural space as an alternative site for clinical islet transplantation in the treatment of type 1 diabetes.

1. Introduction

Type 1 diabetes mellitus (T1D) is an autoimmune disease, in which the insulin-producing β cells of the pancreas are destroyed, resulting in an inability to effectively monitor and regulate blood glucose levels. Approximately 1.2–3.0 million children and adults in the US suffer from T1D and associated healthcare costs exceed $15 billion annually ¹. Since its first discovery and clinical use in 1920, exogenous insulin has been the standard of care and changed T1D from a universally fatal disease to a chronic illness. Due to the inability of exogenous insulin therapy to achieve euglycemia, β cell replacement by islet transplantation has emerged as a promising minimally invasive alternative therapy for T1D ^2,3. Recently, studies in the US by the NIH sponsored Clinical Islet Transplant (CIT) Consortium have demonstrated the safety and effectiveness of the procedure using the intraportal approach and lay the groundwork for Biologic License Application through the FDA to have islets become a reimbursable cell-based therapy for patients with severe T1D. However, the CIT trials also demonstrated that multiple doses of allogeneic islets are generally needed to gain insulin-free normoglycemia due to the inefficiencies of this site ^4,5 that can result in loss of as much as half of the transplanted β cell mass ⁶. Although the precise mechanisms if islet loss are yet to be defined, a role for both a powerful instant blood-mediated inflammatory reaction (IBMIR) to the islets and the hypoxemic environment in the portal venous circulation have been implicated.

Our pilot study evaluated the potential suitability of the oxygen rich intrapleural space⁷ for islet transplantation to successfully achieve safe and physiologic regulation of blood glucose homeostasis, and provides a foundation for more extensive studies to determine the optimal dose and monitoring.

2. Materials and Methods

Further information is available in Supplemental Materials and Methods.

2.1. Animals and Pairing Selection

Donor and recipient were paired based on ABO blood group compatibility and MHC mismatching (Supplementary Fig. 1C). In this pilot study, one donor and one recipient were entered for the study.

2.2. Treatment Regimen

The recipient animal received the therapy regimen as outlined in Fig. 1 A. The day of allogeneic islet transplant is defined as day 0. The treatment regimen included: (1) Thymoglobulin (Anti-thymocyte Globulin-Rabbit, Genzyme, Cambridge, MA) I.V. 5.0 mg/kg on day −3 and day 0, day+5 and day +10; (2) Rituxan (Anti-CD20) I.V. 375mg/M² on day 0 and day +5; (3) Rapamycin (LC Laboratories, Woburn, MA) at an initial dose of 0.2mg/Kg I.M., daily for 3 months total, starting on day −3 adjusted for target blood trough level of 10–20 ng/ml. Islets were infused via a 14 gauge catheter to the right side of pleural space (Fig. 1, B3). Per our standard management for both human and NHP islet transplants, low dose daily insulin (2–4 units) was administrated for the first 28 days post-transplant to promote islet engraftment by facilitating islet “rest”.

Fig. 1. Treatment plan and islet transplantation into intrapleural space.

Panel A: Immunosuppression regimen: The recipient cynomolgus macaque was given: (1) Thymoglobulin(rATG), (2) Anti-CD20 antibody, and (3) Rapamycin (Rapa). Panel B: Donor pancreas was perfused with enzyme (B1) to isolate Islets(B2), which were then transplanted into the right side of intrapleural space via a 14-gauge catheter (B3).

2.3. Diabetes Induction and Management

The recipient NHP received Streptozotocin (STZ) I.V. at dose of 75 mg/kg (Zanosar, Teva Parenteral Medicines, Irvine, CA). Thereafter, blood glucose (BG) levels were monitored twice daily via tail pricking (Accu-check Aviva, Roche Diagnostics, Indianapolis, IN). Diabetes was defined as three consecutive fasting BG readings >300 mg/dL and c-peptide levels < 0.5 ng/mL (Mercodia C-peptide ELISA, Mercodia AB, Uppsala, Sweden). The post-STZ period is defined as from the time of STZ injection until the day of islet transplantation. Post-transplant graft rejection was defined as three days of consecutive fasting BG >180 mg/dL or non-fasting BG >250 mg/dL. Insulin (Humulin R, Lilly, Indianapolis, IN) and Lantus (Lilly, Indianapolis, IN) was administered by sliding scale to achieve BG <250 mg/dL pre-transplant or after graft rejection was defined.

2.4. Islet Isolation and Transplantation

The protocol of islet isolation was based on a modified human islet isolation as previous reported ⁸. Briefly, the pancreas was enzymatically digested using purified Thermolysin and Collagenase blend (Vitacyte, Indianapolis, IN). Islets were purified from the digestion using continuous Optiprep gradient (densities 1.11 to 1.06) and COBE 2991 blood cell processor (Gambro BCT, Inc, Lakewood, CO) to separate islets from exocrine tissue. Final islet preparations were enumerated by manual counting, sizing, and converting islet particle number (IPN) to islet equivalents (IEQ) based on a 150-mm diameter. Islets were then put into 3 mL CMRL 1066 transplant media (Cellgro, Manassas, VA), supplemented with 10% human serum albumin (Grifols Therapeutics, Research Triangle Park, NC) and heparin (70 units/kg of recipient body weight) when ready for transplantation.

Under general anesthesia, a ~3 cm right sided skin incision was performed in an anterolateral position in the 6–7 intercostal space though which a 14-gauge plastic catheter is inserted to access the pleural cavity. The islet preparation was injected through the catheter and the incision closed in layers with absorbable suture.

2.5. Statistical Analysis

Data were not analyzed using statistical models.

3. Results

3.1. Allogeneic Islet Engraftment in Pleural Space Effectively Treats Diabetes

Two weeks after induction of diabetes, a 4.3kg male cynomolgus macaque was transplanted with 0.25ml of packed cell volume tissue that consisted of islets at 15,500 IEQ/Kg body weight with 93% viability and ~85% purity to the right pleural cavity via the 6–7 intercostal space (Fig. 1B). There were no respiratory complications observed during surgery or in the perioperative period. Post operatively, the animal recovered uneventfully and rapidly achieved glycemic control. Fig. 2A shows both pre and posttransplant random non-fasting daily blood glucose (BG) levels and the total daily exogenous insulin requirement. Before transplant, animal glucose levels averaged above 300 mg/dL and received, on average, greater than 15 units of exogenous insulin per day, with occasional needs as high as 23 units of insulin per day. Posttransplant, the animal non-fasting BG readings remained normoglycemic (ranged 56–200mg/dL) during the study duration, supported with 1–3 units of insulin administration per day to prevent post-prandial blood glucose fluctuation. Since we did not measure the fasting BG regularly, it is unclear if the animal can consistently achieve the fasting BG range(30–110mg/dL) of a naïve healthy cynomolgus monkey⁹. Intravenous glucose tolerance test (IVGTT) performed at 28 days and at 92 days post-transplant showed excellence glucose disposal, especially at 92 days, mimicking a normal healthy NHP (Fig. 2B). Both fasting and stimulated insulin and C-peptide (30 minutes after IV glucose) were measured longitudinally throughout study. No measurable insulin or C-peptide was detected in the blood after STZ administration, prior to transplant. Post-transplant, the animal manifest restored insulin stability and C-peptide levels comparable to those of pre-transplant when the animal was healthy and naïve (Fig. 2C). We observed an initial minor weight loss immediately following surgery, followed by continual weight gain (Fig. 2D). Hgb and Hct remained stable over the course of study (Supplementary Fig. 1A), indicating that post-transplant euglycemia was not due to malnutrition.

Fig. 2. Islet transplant into pleural space restored glycemic control in diabetic NHP.

A: Pre and posttransplant non-fasting blood glucose (BG) reading and insulin (INS) usage: Both pre- and post-transplant daily non-fasting BG readings (blue line; left axis) and daily total external INS requirement (red line, right axis) are indicated. B: Intravenous glucose tolerance test (IVGTT): BG disposal dynamics during IVGTT performed at naïve (pre-STZ), post-STZ (After STZ induction but before time of islet transplantation), 28-day and 92-day post-transplant time points. C: Fasting(F) and stimulated(S) insulin and C-peptide. Insulin (left axis) and C-peptide (right axis) levels in serum for the recipient under fasting and post-stimulation at naïve(pre-STZ), Post STZ, 28-day and 92-day post-transplantation. D: Body weight dynamics during the study.

Animal was sacrificed on day 92 post transplantation per study design. Pleural space examination at autopsy showed scattered islet masses on the surface of the parietal pleura overlaying the right hemidiaphragm (Fig. 3A); we did not observe adherence of the lung to the chest wall or diaphragm, and the visceral pleural surface remained grossly smooth. Histology revealed well-preserved islets with strong insulin staining (Fig. 3B), and no CD3 cell infiltration (Fig. 3C), but scattered FoxP3+ cells within the islet mass (Fig. 3D). Histology of lung adjacent to the surgical location showed normal morphology (not shown). The native pancreas was devoid of islet structures (Fig. 3E) and insulin staining (Fig. 3F). These data suggest that islets transplanted to the intrapleural space survived and enabled glycemic control, establishing the pleural space as a feasible and safe transplant site for allogeneic islets in NHP.

Fig. 3. Islet graft histology, immunochemistry profiles and absence of alloantibody responses in blood circulation.

Necropsy graft samples were collected, and sections were stained with H&E, insulin, CD3, FoxP3 (brown). A: Pleural examination at autopsy on day 92 post transplantation showed scattered islets masses growing on the surface of pleural area that layered over the right side of diaphragm. B-D: Insulin (B), CD3(C) and FoxP3 (D) staining within transplanted islets. E-F: The native pancreas was devoid of islet structures (E) and insulin staining (F). G: Serum samples were incubated with donor PBMCs, the degree of IgG binding to either MHC I or MHC II was analyzed by flow cytometry to detect alloantibody response. MFI fold increase of binding to MHC I (right axis) and MHC II (left axis) at day 56 and 92 post-transplant vs naïve.

3.2. Effective Systemic Immunosuppression Protect Graft Survival

We examined lymphocyte subsets by flow cytometry pre-transplantation and at multiple pre-specified time points post-transplant. The conventional immunosuppression protocol (Fig. 1A) utilized (Thymo and Rituxan induction, with maintenance Rapamycin) effectively depleted both T and B cells in circulation (Fig. 4G). The absence of evidence of graft rejection, together with the immune profiling data, suggests our adopted immunosuppression protocol successfully suppressed effector responses against the graft, protected and promoted islets survival in this setting. Further immune profile analysis is available in Supplemental Materials and Methods.

Fig. 4. Systemic immunosuppression effectively diminishes B and T cell populations.

Immune population dynamics in peripheral blood were assessed. WBC and lymphocyte absolute counts (A); CD3+CD4+ and CD3+CD8+ cells (B); CD20+cells (C); CD4+ naïve T cells (Tn), effector memory cells (EM), and central memory (CM) cells (D); CD8+ naïve (Tn), effector memory (EM), and central memory (CM) cells (E); and CD20+CD21+IgM+ and CD20+CD21-CD27+ cells (G) at Pre-Tx(naïve), and time points post transplantation.

4. Discussion

Intraportal administration of pancreatic islets has been the favored strategy for islet transplantation in clinical practice¹⁰, primarily because it has been the only consistently successful approach. Using this technique, islets are administered intravenously into the portal system, where they lodge in the liver capillary bed ^4,11. However, contact with blood induces IBMIR, resulting in loss of approximately 50% of the transplanted β cell mass ^6,12,13, contributing to the common requirement for multiple transplants from different donors to gain a sufficient engraftment mass to secure euglycemia^14,15. Additionally, introduction into the portal system exposes the islets to a hypoxemic environment, which is not alleviated until the graft becomes vascularized ¹⁶. Intrahepatic transplantation also limits the tissue volume that can be transplanted and exposes islets to toxic levels of immunosuppressants in the portal circulation. In addition, the portal site has been associated with potentially severe complications, including bleeding and portal vein thrombosis ^17,18. Efforts have been made to identify alternative sites that may provide a more hospitable environment. These include intrathymic¹⁹, anterior chamber of the eye²⁰, testicle²¹, intracranial²², bone marrow²³, intramuscular²⁴, omental^25,26, subcutaneous²⁷ and gastric submucosa²⁸. While these sites possess theoretical advantages over the liver as a site for implantation, these locations are hampered by poor oxygenation and inconsistent outcomes in rodent and large animal islet transplantation models.

Experimental data suggest that it takes about two weeks for transplanted islets to re-establish vascularization ^29,30. Thus, the early survival of the islets relies on passive diffusion from surrounding tissue. An ideal islet transplantation site should therefore provide optimal oxygenation of the islets in the immediate transplantation period and also provide access to healthy capillary beds to allow for the delivery of insulin into the portal or systemic circulation. The intrapleural space was first proposed two decades ago by Vacanti et al., as an attractive site for engraftment of highly metabolically active engineered tissues, such as hepatocytes or pancreatic islets, because of a significantly higher oxygen tension compared to the intra-peritoneal or other spaces ⁷. However, as far as we are aware, the site has never been formally tested until the studies reported herein.

Vacanti et al. demonstrated that the pO2 of saline instilled into the pleural space was nearly three-fold higher than saline instilled into the peritoneum (188.9 mmHg vs. 68.0 mmHg) at FiO2 of 0.25. Furthermore, the pO2 in the intrapleural space increases with increasing FiO2, while the intraperitoneal pO2 remains constant over a range of super-physiologic FiO2. These data indicate that the intrapleural pO2 is a consequence of direct diffusion of O2 from the lung parenchyma into the pleural space, while oxygenation of the intraperitoneal location is primarily driven by venous blood flow ⁷. These results suggested that, as predicted by Vacanti et al (7), intrapleural injection of islet cells may benefit from markedly increased levels of O2 diffusion and that the level of oxygen supply can be augmented through a simple increase in FiO2 to encourage cell survival in the immediate post-transplantation setting.

In this pilot study, we were only able to transplant a dose of islets (15,500 IEQ/kg) of recipient body weight (RBW) due to the use of a relatively small donor. In our previously reported experience with intrahepatic islet transplantation in NHP, a therapeutic dose of ~25,000 IEQ kg/RBW was required for full insulin-free robust normoglycemic ⁸. However, in the case reported here, despite the islet dose being markedly lower than our previous intrahepatic islet transplant studies, euglycemia was achieved in vivo and IVGTT revealed robust glycemic control at both day-28 and day-92 post-transplant, comparable to the glucose disposal dynamics when the animal was naïve. Fasting and stimulated blood insulin and C-peptide levels were not measurable after diabetes induction, but at day-28 and day-92 post-transplant were comparable to that of the naïve animal (Figure 2. C). These functional data suggest the pleural space provided an efficient site for islet engraftment and function. The noteworthy efficiency may be due to: 1) a more optimal environment for the initial transplant engraftment propelled by higher pO2, related to direct diffusion of O₂ from the lung parenchyma; 2) reduced initial graft tissue loss because of the lack of IBMIR; and/or 3) reduced graft exposure to toxic levels of immunosuppressants.

The pleural space is also easily accessible by minimally invasive approaches, and we safely performed the transplantation without any surgical complications such as bleeding and pneumothorax. We did not notice any development of adherence of the lung to the chest wall or diaphragm, and no grossly evident changes to the adjacent lung at necropsy in our study. Also, we noticed islets engrafted on the surface of parietal pleura overlaying the right hemidiaphragm rather than visceral pleura. We speculate this might simply be due to gravity. At the time of transplant, when islets were injected with solution, they naturally fell to the most dependent position in the chest cavity, which is the diaphragmatic surface for a mostly upright anima like NHP. Although we did not perform a biopsy of the implanted islets in our study, it is presumed that a biopsy could be easily conducted to monitor graft survival in the intrapleural space by thoracoscopy since the grafts can be easily identified, as shown in Fig. 3A. In addition to the advantages mentioned above, compared to the intraportal approach, the pleural space can accommodate more transplant tissue volume, allow for multiple transplantations, and avoid the risk of portal vein bleeding or portal vein thrombosis due to islet placement. Theoretical complications that could accompany transplantation to the pleural space include bleeding, pneumothorax and development of adhesions; however, these should be very rare. While in the current study, the animal non-fasting BG readings ranged 56–200mg/dL during the posttransplant study duration, we could not know if the animal can consistently achieve the fasting BG range(30–110mg/dL) of a naïve healthy cynomolgus monkey since we did not measure the fasting BG regularly. Also, importantly, in the current study, the preparation of NHP islets employed was relatively pure (~85%) whereas human preparations may contain as much as 70% exocrine tissue that could induce both local inflammation in the lung and degradation of islets due to enzyme release in closed space. This concern, however, may be avoided completely in the future when highly pure stem cell-derived islet products become available.

In summary, we provide proof-of-principle that intrapleural islet transplantation can be a safe and effective approach to reversal of diabetes by islet transplantation in a pre-clinical NHP model. Given the attributes of the intrapleural space including the absence of IBMIR, higher oxygen tension, feasibility for multiple infusions and less restriction on transplanted tissue volume, our preliminary report forms the foundation for additional comprehensive studies to determine the optimal dose and monitoring. Prompted by the insightful ideas of Vacanti et al more than 20 years ago (7), the current studies document the potential for hospitable residence of metabolically tissue in the pleural space.

Abbreviations:

(IPS)

Intrapleural Space

(NHP)

Non -Human Primate

8. Data Availability:

The data that support the findings of this study are available from the corresponding author upon reasonable request.