Supplementary Methods

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Nathan Colin Norman
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1 Supplementary Methods Non-human primates: recipient pancreatectomy and care Nine captive bred adolescent rhesus macaques (Macaca mulatta) were used as xenograft recipients. As a mode of establishing diabetes, a duodenal-sparing total pancreatectomy was performed at least 2-4 weeks before the intended xenotransplant, as previously described 1. Post-pancreatectomy care consisted of measurement of daily fasting and post-prandial blood sugars, pancreatic enzyme replacement, and nutritional support. Furthermore, all recipients underwent thorough weekly to biweekly physical examinations under conscious sedation by the Yerkes Chief Veterinarian where the extremities were inspected for signs of inflammation, edema, redness, or tenderness. All procedures with regard to non-human primate care were given IUCAC approval and performed in accordance with Yerkes Primate Research Center guidelines and regulations. Pigs and neonatal porcine islet preparation. Either Duroc or Large White crossbreed pigs (Swine Research and Technology Centre, University of Alberta) were used as pancreas donors. Donor pancreata were obtained from 1-2 day old neonatal pigs of either sex (total body weight kg). The neonatal porcine islets were isolated at the Surgical-Medical Research Institute at the University of Alberta, Edmonton, using a previously described modified Korbutt technique 2. Briefly, the neonatal pigs were anesthetized under halothane and subjected to laparotomy and exsanguination. The pancreas was removed, placed in

2 Hanks balanced salt solution (HBSS) supplemented with 0.25% bovine serum albumin (BSA, Sigma, St. Louis, MO), then cut into 1-2 mm pieces and digested with 1.0 mg/ml collagenase (Type XI, Sigma). After filtration through a nylon screen (500 µm), the tissue was cultured at 37ºC in supplemented Ham s F10 medium (Gibco, Burlington, Ontario, Canada) containing 10 mmol/1 glucose, 50 µmol/l isobutylmethlxanthine, 0.5% BSA, 2 mmol/l L-glutamine, 10 mmol/l nicotinamide, 100 U/ml penicillin, and 100 µg/ml streptomycin, for a period of 5 days prior to shipment to Emory s Animal Cell & Tissue Laboratory, where the islet suspensions were cultured overnight and then transplanted into non-human primates on day 7 post-isolation. Pre-transplant characterization of neonatal porcine islets. On the day of transplantation, the islets suspensions were recovered from the culture dishes and washed in Ham s F10 medium. The porcine islets were assessed for islet quantity, viability by SYTOX Green Fluorescent Nucleic Acid Stain (Invitrogen; Eugene, OR), bacterial contamination by both gram stain and culture, and in vitro functional assessment by static incubation assay (Supplementary Table 1). Samples of the final islet preparation were stained with dithizone (Sigma-Aldrich; St. Louis, MO), and the preparation was assessed by counting the number of islets in each of the following size ranges: , , , , , , and μm. The data were mathematically converted to determine the number of islets with an average of 150 μm and were expressed as islet equivalents (IEQ). In vitro glucose stimulation indices (GSI) were calculated from the static incubation

3 assay results by dividing the amount of insulin released at high glucose stimulation concentrations (16.7 mmol/liter glucose) by that released at low glucose concentrations (3.3 mmol/liter glucose), as previously described 2. Intrahepatic xenoislet transplantation. Following receipt, overnight culture, washing, and IEQ counts, the transplant preparations were resuspended in 20 ml of transplant media supplemented with 200 units of heparin and etanercept 3mg/kg (Enbrel; Amgen & Wyeth, Philadelphia, PA). Approximately 6.2 x 10 6 β cells/kg (~50,000 IEQ/kg) of neonatal porcine islets were transplanted intraportally into 8 of the non-human primate recipients (as an exception, one received ~34,000 IEQ/kg), via gravity drainage of the xenoislet preparation into a mesocolic vein through a 22-gauge intravenous catheter. The xenograft recipient that received ~34,000 IEQ/kg was our first xenotransplant and based on this recipient s initial graft function we decided to increase our islet mass to 50,000 IEQ/kg in attempts to obtain the optimal xenograft function. Post-transplantation monitoring of xenograft function. Fasting and postprandial blood glucose levels were monitored (Glucometer Elite; Bayer, Elkhart, IN) daily in non-human primate recipients by way of ear-stick. Insulin (NPH, Ultralente; Eli Lilly, Indianapolis, IN) was administered three times daily to maintain fasting blood glucose at <300 mg/dl. Intravenous glucose tolerance tests (IVGTTs) were performed once before the transplant as a control, as well as at monthly intervals during the post-transplant period. Each IVGTT consisted of

4 obtaining a baseline blood sample, then giving an intravenous bolus of dextrose (500 mg/kg) via the saphenous vein and collecting blood at the 3, 6, 10, and 25-minute or 10, 30, 60, and 90-minute intervals following the bolus, to test sera for changes in glucose, porcine insulin, and porcine c-peptide levels. In addition, serial porcine c- peptide levels were assessed throughout the post-transplant period, using the manufacturer s protocol from Linco s radioimmune assay kit (Linco Research; St. Charles, MO). Briefly, 100 μl of each sample, standard, and quality control were placed in non-specific binding tubes in duplicate. A 100 μl of porcine c-peptide antibody was added and the tubes were incubated overnight at 4 0 C for 48hrs with addition of 125 I-Porcine c-peptide at 24hrs. Then 1.0 ml of cold (4 C) precipitating reagent was added and peptide-antibody precipitates were obtained by centrifugation. All precipitates were counted in a gamma counter and compared against a standard curve to determine the porcine c-peptide concentration. Experimental groups and immunosuppressive and animal treatment protocols. A control group consisting of two animals received islet xenografts, but no immunosuppressive therapy. Seven primates received a costimulation blockade-based immunosuppressive regimen that was begun concomitant with xenotransplantation, consisting of induction therapy with two doses of anti-il-2 receptor antibody (basiliximab) and six doses of anti-cd154 antibody, given over a 2-week period. In addition, these animals received ongoing maintenance therapy with belatacept and sirolimus (Supplementary Fig. 1). One animal (RRf-9) required conversion from sirolimus maintenance therapy to mycophenolate mofetil (14mg/kg twice a day) at post-

5 transplant day 23, secondary to an inability to achieve the therapeutic target levels of sirolimus. Our animal support protocol consisted of: measurement of daily fasting and post-prandial blood sugar, twice daily replacement of pancreatic enzyme with pancrelipase enteric coated microspheres (Creon20; Ortho-McNeil, Raritan, NJ), and daily administration of megestrol acetate (Megace; Bristol-Myers Squibb, Princeton, NJ). Due to problems with weight loss and viral reactivations, a second support protocol was created. This modified animal care support protocol consisted of Creon20 three times a day and discontinuation of Megace on post-transplant day 30, plus the addition of viral prophylaxis with a daily dose of 6 mg/kg of oral valganciclovir (Valcyte; Roche Nutley, N.J) for the length of survival of the recipient. Drug dosages and scheduling used: belatacept was administered intra-operatively (20 mg/kg i.v.) and on postoperative days (POD) 2 and 6. Additional doses were given on POD 14 and every 2 weeks thereafter. The drug was well tolerated, with no evidence of gross toxicity. Anti-CD154 antibody (H106) was administered intra-operatively (20mg/kg i.v.) and on POD 2, 6. 9, 12, and 14. Anti-IL-2 receptor antibody was administered intra-operatively (0.3mg/kg i.v.) and on POD 2. The belatacept and H106 used in these experiments were provided by Bristol-Myers Squibb (Princeton, NJ). Sirolimus (Rapamune), anti-il-2r antibody, and mycophenylate mofetil (Cellcept) were purchased from the Emory University Hospital Pharmacy. Anti-Gal IgG and total anti-pig IgG assays. Serum anti-galα1-3galβ1-4glcnac epitope IgG levels were assessed by enzymelinked immunosorbent assay (ELISA), as previously described 3. Briefly, ELISA plates

6 were coated with 10 μg/ml of gal antigen (V-LABS; Covington, La) linked to bovine serum albumin (BSA). Plates were then blocked with 1% BSA and then incubated with diluted serum samples. After washing, the plates were coated with peroxidase conjugated anti-rhesus IgG (Sigma-Aldrich; St. Louis, Mo) and developed with o-phenylene diamine. Absorbance was measured at 450 nm. In addition to the test plates that were coated with the trisaccharide, a parallel set of background plates coated with buffer only were used in the assay in an identical fashion. Results are expressed as a standardized value by subtracting the absorbance of the 1:2 dilution of the test serum from the absorbance on the background plate. Total anti-pig IgG levels were determined by FACS analysis, using porcine lymphocytes as target cells. Lymphocytes were isolated from peripheral blood, and 100 μl cell suspensions containing 5 x 10 5 lymphocytes were mixed with 100 μl of 1:10 rhesus sera and incubated for 30 min at 4 C. Cells were incubated with media to serve as a negative control. After incubation, the cells were washed twice with PBS and then incubated for 30 min at 4 C with either 50 μl of 1:10 PE-goat antihuman IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA). After incubation, the cells were washed twice with FACS buffer. Cells were then analyzed on a Becton Dickinson FACScan Flow Cytometer. The median fluorescence intensity (MFI) of the cells for each test serum was compared with that produced from the controls. Histological and immunohistochemical evaluation. At time of recipient sacrifice, liver specimens were either fixed in 10% formalin and processed in paraffin blocks for use in standard hematoxylin and eosin histology, or

7 fresh unfixed tissue was frozen in OCT compound (Tissue-Tek, Hatfield, PA) to test for presence of insulin and types of immune cell infiltrates by immunohistochemistry. Specific antigens in frozen tissue sections were labeled with primary antibody, then visualized using the DAKO LSAB + labeled Streptavidin-Biotin kit (Dako; Carpenteria, Ca). The antigen-specific primary antibodies used in these immunohistochemical tests were for insulin (Fitzgerald; Concord, Ma), CD3 (Dako; Carpenteria, Ca), CD4 (BD; Franklin Lakes, NJ), CD8 (Dako; Carpenteria, Ca), CD20cy (Dako; Carpenteria, Ca), CD68 (Dako; Carpenteria, Ca), C4d (Quidel; San Diego, Ca) and neutrophil elastase (Dako; Carpenteria, Ca). In addition, all terminated recipients underwent necropsy by the Yerkes Veterinarian pathologist and all final pathology reports were reviewed for any gross evidence of thrombosis. Xenosis and viral reactivation monitoring. Monitoring for the presence of selected viruses (PERV, RhCMV, and SV40) was done by weekly testing of recipient blood samples by real-time PCR using TaqMan chemistry and reagents from Applied Biosystems. Reactions of 50 μl total volume contained 25 μl TaqMan Universal Master Mix, 7.5 μl genomic DNA isolated from whole blood, and varying concentrations of primers and probe. Amplification and analyses were done on a model 7900HT Sequence Detection System (Applied Biosystems). Our real-time PCR PERV assay was a modification of that published by Switzer et al. 4 PCR primers, from a conserved region of the PERV gag gene that amplifies PERV subgroups A, B, and C, were used with a TaqMan Probe of our design (Primer

8 Express software) to amplify genomic DNA from rhesus whole blood. As a positive DNA control, samples were also amplified with rhesus gamma globin primers; these primer sequences are the same as published by Lobashevsky et al. 5 The PERV forward primer used is (CGG CAA GAG AAG AAT TTG ACT AAG ATC), the reverse primer is (CAG TTC CTT GCC CAG TGT CCT CTT), and the TaqMan probe is (6FAM-TGG CCG CAG TGG TTG AAG GGA A- TAMRA). The RhCMV primers and probe used were as published by Kaur et al. 6 and amplified a 108 bp sequence. The RhCMV forward primer used is (GTT TAG GGA ACC GCC ATT CTG), the reverse primer is (GTA TCC GCG TTC CAA TGC A), and the TaqMan probe is (6FAM-TCC AGC CTC CAT AGC CGG GAA GG-TAMRA). The SV40 primers and probe sequences used are modifications of those published by Shi et al. 7 and amplify a 130 bp sequence from the large T antigen gene. This SV40 forward primer is (TGG AGG AGT AGA ATG TTG AGA GTC A), the reverse primer is (GAT TCC AAC CTA TGG AAC TGA TGA), and the TaqMan Probe is (6FAM-TGG GAG CAG TGG TGG AAT GCC TTT-TAMRA).

9 Reference: 1. Adams, A. B. et al. Diabetes 51, (2002). 2. Korbutt, G. S. et al. J Clin Invest 97, (1996). 3. Richards, A. C. et al. Transplantation 73, (2002). 4. Switzer, W. M. et al. Transplantation 68, (1999). 5. Lobashevsky, A. et al. Tissue Antigens 54, (1999). 6. Mueller, N. J. et al. J Virol 76, (2002). 7. Shi, L., Ho, J. et al. Biologicals 27, (1999).

Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.

Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification. Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA