Successful Reversal of Streptozotocin-Induced Diabetes with Stable Allogeneic Islet Function in a Preclinical Model of Type 1 Diabetes Judith M
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Rapid Publication Successful Reversal of Streptozotocin-Induced Diabetes With Stable Allogeneic Islet Function in a Preclinical Model of Type 1 Diabetes Judith M. Thomas,1 Juan L. Contreras,1 Cheryl A. Smyth,1 Andrew Lobashevsky,1 Stacie Jenkins,1 William J. Hubbard,1 Devin E. Eckhoff,1 Scott Stavrou,2 David M. Neville Jr.,2 and Francis T. Thomas1 The recent focus on islet transplantation as primary than a deletional process underlies this operational therapy for type 1 diabetes has heightened interest in tolerance model. This study provides the first evidence the reversal of type 1 diabetes in preclinical models us- that operational tolerance can protect MHC nonhuman ing minimal immunosuppression. Here, we demonstrat- primate islets from rejection as well as loss of func- ed in a preclinical rhesus model a consistent reversal of tional islet mass. Such an approach has potential to all measured glycemic patterns of streptozotocin-induced optimize individual recipient recovery from diabetes as type 1 diabetes. The model used single-donor islet trans- well as permitting more widespread islet transplanta- plantation with induction of operational tolerance. The tion with the limited supply of donor islets. Diabetes 50: term “operational tolerance” is used to indicate durable 1227–1236, 2001 survival of single-donor major histocompatibility com- plex (MHC)-mismatched islet allografts without main- tenance immunosuppressive therapy and without rejec- tion or loss of functional islet mass or insulin secretory solated pancreas islet allotransplantation (IPIT) has reserve. In this operational tolerance model, all immu- attracted recent attention as an imminently promis- nosuppression was discontinued after day 14 posttrans- ing approach to achieve euglycemia and long-term plant, and recipients recovered with excellent health. relief from exogenous insulin therapy in patients The operational tolerance induction protocol combined I with type 1 diabetes. Shapiro et al. (1) established proof of peritransplant anti-CD3 immunotoxin to deplete T-cells principle in a systematic series of seven type 1 diabetic and 15-deoxyspergualin to arrest proinflammatory cyto- patients at the University of Edmonton, Edmonton, Al- kine production and maturation of dendritic cells. T-cell berta. This group has now been expanded to 13 patients, deficiency was specific but temporary, in that T-cell– dependent responses in long-term survivors recovered the large majority of whom are insulin-independent at 1 to normal, and there was no evidence of increased sus- year (Rajotte R, personal communication). Notably, pro- ceptibility to infection. Anti-donor mixed lymphocyte longed reversal of type 1 diabetes with novel immunosup- reaction responses were positive in the long-term survi- pressive and tolerogenic strategies has also been reported vors, but all showed clear evidence of systemic T-helper after isolated islet transplantation in four nonhuman pri- 2 deviation, suggesting that an immunoregulatory rather mate series (2–5). Kenyon et al. (3,4) reported long-term survival in seven of seven baboon and six of six rhesus monkey recipients using monthly maintenance immuno- From the 1Division of Transplantation, Department of Surgery, University of suppressive therapy with anti-CD154 (hum58Biogen). An- Alabama Medical Center, Birmingham, Alabama; and the 2National Institutes other study from our group reported rejection-free long- of Health, National Institute of Mental Health, Laboratory of Molecular Biology, Bethesda, Maryland. term survivors at 3 months to Ն1 year without any Address correspondence and reprint requests to Judith M. Thomas, Suite maintenance immunosuppressive therapy after concor- 563, Boshell Diabetes Research and Education Building, The University of Alabama at Birmingham, 1808 Seventh Ave. S., Birmingham, Alabama 35294. dant islet xenotransplantation and brief anti-CD3 immu- E-mail: [email protected]. notoxin (IT) and cyclosporine treatment in three of three Received for publication 25 January 2001 and accepted in revised form 23 spontaneously diabetic nonhuman primates (5,6). Most March 2001. Posted on the World Wide Web at www.diabetes.org/diabetes on 4 May 2001. importantly, these primates showed durable and un- changed functional islet mass and normal acute insulin J.M.T. has received consulting fees from Novatris. release (6). BG, blood glucose; DSG, 15-deoxyspergualin; ELISA, enzyme-linked immu- noassay; FBS, fetal bovine serum; HBV, hepatitis B virus; IEQ, islet equivalent; In concert, these advances generated momentum for ␥ ␥ IEQ150, IEQ normalized to 150- mol/l diameter; IFN , -interferon; IL, inter- islet allotransplantation as a primary therapy for human leukin; IPIT, isolated pancreas islet transplant; IT, immunotoxin; IVGTT, type 1 diabetes. However, there are drawbacks to consider. intravenous glucose tolerance test; Kg, glucose disposal rate; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; NF, nuclear There is an uncertain risk that allogeneic islet transplant factor; R-D, recipient-donor; PBL, peripheral blood lymphocyte; PCR, poly- recipients will undergo recurrent autoimmune disease (7). merase chain reaction; PHA, phytohemagglutinin; RR, relative response; SI, stimulation index; SSP, sequence-specific primer; STZ, streptozotocin; TH, Additionally, there are complications of maintenance im- T-helper. munosuppressive therapy to control rejection, and there DIABETES, VOL. 50, JUNE 2001 1227 REVERSAL OF STZ-INDUCED DIABETES WITH STABLE ALLOGENEIC ISLET FUNCTION are requirements for retransplantation to offset loss of Laboratory Animals (16) and were approved by the institutional animal care functional islet mass and insulin secretory reserve that and use committee. Restraint for bleeding and intravenous glucose tolerance test (IVGTT) occurs in both early and late follow-up after IPIT (1,8–11). procedures was achieved with an intramuscular injection of 10 mg/kg ket- To date, there have been no published reports of long-term amine (Fort Dodge Laboratories, Fort Dodge, IA) mixed with 1 mg/kg function of single-donor major histocompatibility complex acepromazine (Vedco Laboratories, St. Joseph, MO). Antibiotic treatment was (MHC)-incompatible allogeneic IPIT for Ն1 year without cephazolin (Eli Lilly, Indianapolis, IN) given at 12.5 mg/kg i.m. twice daily on maintenance immunosuppressive therapy and/or exoge- days 0 to ϩ6 and oral vibramycin (Pfizer, Evanston, IL) given at 1.5 mg/kg on ϩ ϩ ⅐ –1 ⅐ –1 nous insulin in human IPIT recipients, and there has been days 7to 21. Oral Ensure Plus at 15 mg kg day provided supplemental nutritional support for 2 weeks posttransplant. Buprenorphine hydrochloride only one report of operational tolerance in naturally (0.05 mg/kg i.m. every 12 h) (Reckit Colman Pharmaceuticals, Richmond, VA) diabetic nonhuman primate IPIT recipients (5). The was used for analgesia after surgery. healthy rejection-free course and stability of functional Immunosuppressive treatment. IPIT recipients were induced on the day of islet mass in the latter study (6) provided a rationale to transplantation with one of three protocols, two of which used F(Ab)2-IT, a conjugate of IgG or F(Ab) fraction of FN18 anti-rhesus CD3⑀ mAb and CRM9 examine tolerance induction to allogeneic IPIT in an 2 mutant diphtheria toxin. The three protocols included F(Ab) -IT alone, inducible streptozotocin (STZ) preclinical type 1 diabetes 2 F(Ab)2-IT plus DSG, or DSG alone. F(Ab)2-IT was prepared by D.M.N., as nonhuman primate model. described (14). In addition, all IPIT recipients had intravenous methylpred- We recently reported in nonhuman primate recipients of nisolone (Upjohn, Kalmazoo, MI) administered at 7 mg ⅐ kg–1 ⅐ day–1 given 4 h kidney allograft a novel approach to durable chronic pretransplant and at 3.5 mg ⅐ kg–1 ⅐ day–1 and 0.35 mg ⅐ kg–1 ⅐ day–1 on the next 2 days, respectively. The first of two F(Ab) -IT infusions were administered rejection-free tolerance that persists for years without 2 intravenously as a 100 g/kg bolus at 2–3 h pretransplant. For the second maintenance immunosuppressive therapy (12). This strat- ϩ treatment, F(Ab)2-IT at the same dose was infused on day 1. DSG (NKT-01; egy used a short peritransplant treatment combination Bristol Myers, Princeton, NJ, and Nippon Kayaku, Tokyo) was administered at with anti-CD3 diphtheria-based IT. The IT depletes the 2.5 mg ⅐ kg–1 ⅐ day–1 i.v. beginning 4 h pretransplant and continuing through day lymphoid system of circulating and sessile T-cells (13) ϩ14. The DSG was reconstituted in saline, kept at 4°C, and administered of both naı¨ve and memory phenotypes (13a), whereas within 72 h after reconstitution. Intravenous fluids (Abbott Labs, Abbott Park, IL) were administered on days 0–5 to maintain hydration. Other than the islet 15-deoxyspergualin (DSG) concomitantly blocks proin- infusion, the recipients did not receive any blood or other cell transfusions. No flammatory cytokine production and the maturation of den- immunosuppressive agents were given after day 14 posttransplant. Two STZ- dritic cells by inhibiting nuclear translocation of nuclear induced diabetic animals received F(Ab)2-IT plus DSG without IPIT. factor (NF)-B (14,15). The achievement of durable spe- Induction of type 1 diabetes with STZ. To induce type 1 diabetes, normal cific immune tolerance to kidney allografts with docu- recipients were treated 1–4 weeks before IPIT with an intravenous bolus of STZ at 140 mg/kg. The STZ (Sigma, St. Louis, MO) was mixed with 5 ml citrate mented MHC class I and class II incompatible alleles (12) buffer and infused over 1 min. As previously reported, a single large (Ͼ140 led us to postulate that prevention of islet allograft rejec- mg/kg) dose, but not smaller single or multiple STZ doses, reliably induces a tion and maintenance of stable functional islet mass on a type 1 diabetic state that is both similar to human type 1 diabetes and asso- long-term basis might also be achievable without mainte- ciated with similar secondary complications (17,18). nance immunosuppressive therapy using the IT plus DSG By day 3 post-STZ, all animals exhibited hyperglycemia, with fasting blood glucose (BG) Ͼ250 mg/dl.