American Journal of Transplantation 2015; 15: 2739–2749 Wiley Periodicals Inc.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13329
Brief Communication
Pilot Study Evaluating Regulatory T Cell–Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model J. Lei1,*, J. I. Kim1, S. Shi1, X. Zhang1, Z. Machaidze1, S. Lee1, C. Schuetz1, P. N. Martins1, T. Oura1, E. A. Farkash2, I. A. Rosales2, R. N. Smith2, R. Stott1, K. M. Lee1, J. Soohoo1, S. Boskovic1, K. Cappetta1, O. M. Nadazdin1, Y. Yamada1, H. Yeh1, T. Kawai1, D. H. Sachs1, G. Benichou1 and J. F. Markmann1,* 1
Center for Transplantation Science, Massachusetts General Hospital, Harvard Medical School, Boston, MA 2 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA Corresponding authors: James F. Markmann and Ji Lei, [email protected]; [email protected]
The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(30 -dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, antiinterleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donorspecific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.
diabetic complications (3–5). Islet transplantation results have improved with insulin independence rates approaching or equaling those seen in whole organ pancreas transplants at 5 years (6,7). However, current immunosuppressive agents are not only toxic to the recipient but are specifically toxic to beta cells and may induce peripheral insulin resistance (8,9). Because the risks of chronic immunosuppression may exceed the risks of diabetes-related complications for the majority of diabetics, islet transplantation is limited to those with severe labile disease with hypoglycemic unawareness. Thus, the full potential of isolated islet transplantation will not be realized until islet survival can be secured without chronic immunosuppression. A conceptually attractive path to donor-specific tolerance is through delivery of donor antigen in a tolerogenic manner (10). However, a potential pitfall of donor antigen exposure is recipient sensitization. A theoretical solution to this problem is found in classic experiments from Jenkins and Schwartz who determined that 1-ethyl-3-(30 -dimethylaminopropyl)-carbodiimide (ECDI) treatment of stimulators in vitro resulted in donor-specific anergy in responding T cells in mixed lymphocyte reaction-type assays (11). In this study, we examined the translational potential of administering ECDI-treated donor lymphocytes as a means to deliver nonstimulatory and nonsensitizing donor antigen in a tolerogenic manner, an approach that was recently reported to attain islet and heart graft tolerance in mice (12,13).
Materials and Methods
Abbreviations: DLI, donor splenocytes infusion; ECDI, ethylenecarbodiimide; NHP, nonhuman primate
Further information is available in Supplemental Materials and Methods.
Received 26 October 2014, revised 01 March 2015 and accepted for publication 20 March 2015
Animals and pairing selection Donors and recipients were paired on the basis of ABO blood group compatibility and MHC mismatching (Figure S1). In each case, one donor (weight range 5.0–9.2 kg) was used for one recipient (weight range 3.9–6.2 kg).
Introduction Treatment regimen
Isolated pancreatic islet transplantation offers minimally invasive restoration of the beta cell mass in type I diabetes (1,2) with potential for prevention and reversal of
All control and treated recipient animals received therapy consisting of (Figure 1A) the following: (1) thymoglobulin (anti-thymocyte globulin-rabbit, Genzyme, Cambridge, MA) I.V. 10 mg/kg on day 7 and day 6; (2) tocilizumab
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Figure 1: Significant prolongation of allogeneic islet graft survival by ECDI-treated donor splenocyte infusion. (A) Treatment plan. All cynomolgus macaques (nonhuman primates [NHPs]) were given thymoglobulin (thymo) and anti-IL6R. Daily rapamycin (rapa) was administrated for 1 month starting on day 3. Allogeneic islets were transplanted on day 0. ECDI-DLI was delivered via portal vein on the same day along with islet transplantation for experimental group only. (B) Glycemic control was maintained for 133, 48, 80, and >81 days for four ECDI-DLI experiment group NHPs (E1–E4), and 9, 18 days for two control group NHPs (C1, C2). Mean survival time of the ECDI-DLI group 85.5 days versus non-ECDI group 13.5 days, p ¼ 0.0177.
(anti-IL6R, Chugai Pharm, Tokyo, Japan [14]) I.V. at a dose of 10 mg/kg on days 7, 0, 7, 14, 21; (3) rapamycin (LC Laboratories, Woburn, MA) at a dose of 0.2 mg/Kg sc daily for total 1 month starting on day 3 with target blood trough level of 10–20 ng/mL; (4) ECDI-DLI was administered only to experimental group animals on day 0; and (5) allogeneic islet transplant was performed on day 0. Both islets and ECDI-DLI were infused via a branch of the portal vein. Daily low-dose insulin (2–4 units) was administrated for the first 30 days posttransplant to promote islet engraftment by allowing islet ‘‘rest’’ for both control and experimental group animals.
Diabetes induction and management NHPs received streptozotocin 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 180 mg/dL or nonfasting BG >250 mg/dL. Insulin (Humulin R, Lilly, Indianapolis, IN) and Lantus (Lilly) was administered by sliding scale to achieve BG 50% purity by Dithizone stain were combined for transplantation. Final islet preparations were enumerated by manual counting and sizing, and converting islet particle number (IPN) to islet equivalents (IEQ) based on a 150-mm diameter. Islets were then put into 15 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 ready for transplantation. Under general anesthesia, a small midline abdominal incision was performed to cannulate a branch of superior mesentery vein with an 18-gauge catheter
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Tregs Expansion to Protect Islet Graft Survival to access the portal system. The islet preparation was slowly infused by gravity over a period of approximately 10–15 min.
ECDI-treated cell preparation and infusion ECDI-treated donor lymphoid cells were delivered to experimental group recipients intraportally immediately after islet infusion. The protocol for ECDI treatment was previously described (16) and included harvest of spleen and lymph node, processing into single cell suspensions, lysis of RBC’s and incubation of lymphoid cells with ECDI (Merck KGaA [Novabiochem], Darmstadt, Germany) 75 mg/mL per 3.2 108 cells at 48C for 1 h with gentle shaking followed by washing and filtering to remove cell clumps. Prior to I.V. injection, samples of ECDI-treated lymphoid cells were stained for apoptosis marker 7-AAD and annexin V (both eBioscience, San Diego, CA). At each transplant, ECDI-treated cells from a single donor were infused in a volume of 30 mL.
Statistical analysis Data were analyzed using GraphPad Prism (version 5, GraphPad Software). Graft survival between experimental and control groups was compared using Log-rank (Mantel-Cox) Test and Gehan–Breslow–Wilcoxon statistics. Other differences between experimental group and control were analyzed using the Student’s unpaired t-test and F-test. p-Values less than 0.05 were considered statistically significant.
Results Significant prolongation of allogeneic islet graft survival by ECDI-treated donor lymphoid cell infusion A total of six diabetic NHP, four in the ECDI-DLI experimental group (referred to as E1–E4) and two in the non-ECDI-DLI control group (C1 and C2), were transplanted with single donor islets. Details of the characteristics of the recipients and transplants are summarized in Table 1. For the ECDI-DLI experimental group, recipients received between 4.5 108 and 3.75 109 ECDI-treated donor lymphoid cells via portal vein infusion. Prompt and excellent glycemic control was maintained for 133, 48, 80 and >81 days for the experiment group, E1–E4, respectively (mean survival time [MST] ¼ 85.5 days. Figure 1B). In contrast, the control group of NHP which received an identical regimen absent ECDI-DLI maintained glycemic control for 9 and 18 days (MST ¼ 13.5 days, Figure 1B) (ECDI-DLI group vs. non-ECDI-DLI group, p < 0.0177).
In animal E1, ideal BG control persisted for 133 days until loss of graft function (Figure 2A). An intravenous glucose tolerance test (IVGTT) performed at 5 weeks posttransplant showed normal glucose disposal similar to pre-STZ administration (Figure S3A). Liver biopsy performed at 146 days revealed most islets were infiltrated by lymphocytes, consistent with rejection (Figure S4A and B). Animal E2 glycemic control continued for 48 days posttransplant (Figure 2B). IVGTT performed at 5 weeks posttransplant showed a normal BG response (Figure S3B). Glycemic control partially deteriorated at 8 weeks. This recipient demonstrated elevated alkaline phosphatase and lipase (4 upper limit of normal, data not shown), persisting throughout the post-STZ and transplant course. Upon sacrifice at day 62 posttransplant, necropsy revealed occasional well-preserved, insulin-positive islets in the liver, but most islets were infiltrated by lymphocytes, consistent with rejection (Figure S4C1 and data not shown). The most striking finding was disseminated CMV infection evident in kidney (Figure S4C2) and the pancreas. Animal E3 maintained glycemic control posttransplant for 50 days (Figure 2C), then required low-dose (4–6 units) insulin to maintain acceptable nonfasting glucose level. Because of the imperfect graft function, a liver biopsy was performed at day 68. Histology revealed numerous wellgranulated, insulin-positive islets with minimal to no infiltrating lymphocytes (Figure 3A1) suggesting the absence of rejection. We suspect the imperfect glycemic control with eventual hyperglycemia represented marginal mass exhaustion which can occur in NHP isografts (17); however, the lack of lymphocytes infiltration on biopsy could also be due to sampling error. IVGTT performed at 40 and 70 days posttransplant showed normal glucose homeostasis comparable to the pre-STZ period (Figure S3C). Animal E4 achieved glycemic control (Figure 2D) but was sacrificed at day 81 due to palm injury from unclear causes. Autopsy revealed well-preserved islets with no indication of rejection (Figure S4D1 and D2). IVGTT performed at 64 and
Table 1: Summary of donors, recipients, transplanted islets and ECDI-treated donor spleen cells Experimental group Recipient ID Recipient body weight (RBW) (kg) Donor body weight (kg) Donor pancreas weight (g) Islet particle number Number of islet equivalent (IEQ) Pack cell volume (mL) Islet purity (%) Islet viability (%) IEQ/RBW kg Total ECDI-treated donor spleen cell# ECDI-treated donor spleen Cell#/Kg RBW
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3.84 7.5 10.6 146000 125600 0.3 80 98 32 708 4.5 108 1.17 108
5.1 7.0 8.5 113900 103200 0.4 70 99 20 235 1.5 109 2.94 108
6.2 9.2 10.0 132000 107400 0.4 75 95 17 323 3.75 109 6.05 108
4.3 5.0 8.5 121500 112000 0.4 80 99 26 047 N/A N/A
3.9 5.7 8.7 130500 119500 0.4 80 99 30 641 N/A N/A
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Survival Post-transplant(days) Figure 2: Glycemic control posttransplant. Four ECDI-DLI experimental group and two control group diabetic NHPs each promptly achieved normoglycemia postallogeneic islet transplant. Daily low-dose (2–4 units) insulin (INS) was administrated to all experimental and control group animals to help initial islet engrafting for the first 30 days posttransplant. Blood glucose (BG) measurements are nonfasting random daily tests.
81 days posttransplant showed normal glucose homeostasis comparable to the pre-STZ period (Figure S3D). Graft survival is associated with the presence more of CD4þ Foxp3þ T cell in and around grafts To characterize lymphocytic graft infiltration, we performed biopsy when grafts were functional. Biopsy histology of E3 (Figure 3A) revealed numerous well-granulated islets (A1) with strong insulin staining (A2), minimal to no infiltration of CD20þ (A3), CD8þ (A4), or CD4þ (A5) cells into islets. Lymphocytes remain at the periphery to form a cuff around the islets. In contrast, necropsy of hyperglycemic animal E3 revealed disrupted islet architecture (Figure 3B) with diminished insulin staining (B2) at which points we observed intensive CD20þ (B3), CD8þ (B4), and CD4þ (B5) cells infiltrating the islets. Most notably, during normoglycemia, biopsy of grafts revealed that the number of CD4 (A5 brown)/ Foxp3 (A5 blue) positive cells in and around islets was consistently higher than at the time of rejection similar to the 2742
findings in mouse models (B5 blue) (13,18). Biopsy performed on one additional experimental animal (E4, data not shown) at the time of normoglycemia revealed the similar histological findings as E3 during normoglycemia. Necropsy histology assessment of recipient E1 (Figure S4A and B), E2 (Figure S4C), and two control animals C1 and C2 (Figure S5) was also performed when grafts were rejected. The pattern of islet destruction and cell infiltration was consistent among all rejected animals, experiential and control. Significant CD4þCD25þFoxp3þ generation and expansion associated with graft survival post-ECDI-DLI To study the dynamics of Treg levels in the circulation, PBMCs were stained for CD4, CD25, and Foxp3 at pretreatment (naive) and several time points post-ECDI-DLI. We observed a significant relative expansion in the proportion of CD4þCD25þFoxp3þ Tregs in PBMC by flow in all four ECDI-DLI recipients. The peak fold increase ranged from 9- to 21-fold compared to that of pretreatment levels (Figure 4A). American Journal of Transplantation 2015; 15: 2739–2749
Tregs Expansion to Protect Islet Graft Survival
Figure 3: Islet graft histology and immunochemistry. Biopsy and necropsy graft samples were collected and sections were stained with H&E, insulin (brown), CD20, CD8, and CD4 (brown)/Foxp3 (blue). (A) At time of normoglycemia at day 68, biopsy of recipient E3 revealed well-granulated islets (A1) with strong insulin staining (A2), minimal to no infiltration of CD20þ (A3), CD8þ (A4), or CD4þ (A5) cells into islets. (B) Necropsy of grafts taken from hyperglycemia animals revealed ruptured islet structures (B1) with much diminished insulin stain (B2). Intense lymphocytic infiltration into the islets (B3, B4, B5) were observed. Most notably, biopsy of survival grafts revealed that the number of CD4 (brown)/Foxp3 (blue) positive cells (A5) in and around islets are higher compared with rejected grafts (B5). Photomicrographs are representative of five sections of a recipient (E3). The patterns of cell infiltration were consistent across animals studied. Arrows indicate islets. More detailed histology data at Figures S4 and S5.
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The detected Treg average peak of experimental group is significantly higher than that of control group (Figure 4B, p ¼ 0.0256). Peak expansion was detected during the first 1–5 weeks post-ECDI-DLI (Figure 4A). The detailed Treg dynamics of animal E3 (Figure 4C and D) is presented showing that Tregs peaked around day 5 post-ECDI-DLI and Treg levels remained elevated for about 3 weeks at which point levels gradually decreased but stabilized at levels higher than levels pretreatment. Similar trend was observed in all ECDI-DLI recipients. In the control group, neither recipient demonstrated an elevated percentage of Tregs suggesting a critical role of ECDI-DLI in the expansion of Tregs (detailed Treg percentage change and absolute Treg number count for all animals are presented in Figure S6). Lymphocytes from ECDI-DLI recipients exhibit donorspecific hyporesponsiveness To assess for donor-specific hyporesponsiveness, we examined alloreactivity of recipient PBMC by ELISPOT to
donor PBMC and to third party PBMC at multiple time points posttransplant. While normoglycemic (NG), all ECDIDLI recipients demonstrated significantly reduced responses to donor PBMC relative to third party PBMC as measured by IFN-g ELISPOT (Figure 5A–D). Donor-specific hyporesponsiveness was lost when assessed at the time of graft rejection (hyperglycemia, HG). In contrast, lymphocytes from recipients in the control group exhibited no donor-specific hyporesponsiveness during the period of normoglycemia (Figure 5E and F). These data suggest that donor-specific T effectors were rendered at least transiently unresponsive by the ECDI-DLI protocol. Absence of alloantibody response upon ECDI-DLI treatment We examined the development of anti-donor antibody (IgG1 isotype) in two ECDI-DLI recipients by flow cytometry. Animal E4 posttransplant serum samples on days 14 and 64, time points while the graft was still functioning, were analyzed. Flow
Figure 4: Significant Treg expansion in ECDI-DLI recipients. (A) All four ECDI-DLI experimental group diabetic NHPs but not control group demonstrated evidence of Treg expansion in PBMC by flow. The detected peak increase ranged from 9- to 21-fold compared to that of pretreatment (naive). The day of detected peak relative to transplant is shown on the top of each bar graph. (B) The detected average of fold increase of Treg of the experimental group is significantly higher than that of control group, p ¼ 0.0256. (C and D) Detailed flow of Treg levels of recipient E3 are shown from multiple time points. (C) High Treg levels were maintained for about 3 weeks before gradually decreasing in animal E3. More detailed histology data at Figure S6.
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Figure 5: ECDI-DLI induces donor-specific hyporesponsiveness. Responsiveness was assayed by measuring IFN-g production by ELISpot and recipient PBMCs were used as responders. (A–D) All four ECDI-DLI experimental group diabetic NHPs demonstrated evidence of donor-specific hyporesponsiveness during normoglycemia. Recipients PBMCs react remarkably less to donor cells than to third party demonstrated when grafts were functioning. Donor-specific hyporesponsiveness was lost when grafts were rejected. For animal E1 and E2 (A and B), there are no pretreatment (naive) data available. (E and F) Control recipients without ECDI-DLI showed no donor-specific hyporesponsiveness during normoglycemia period of study.
cytometry revealed no alloantibody response at these two time points (Figure 6A). Animal E3 posttransplant serum samples on days 36 and day 86 posttransplant, before and after rejection, were analyzed (Figure 6B). As with E4, E3 exhibited no donorreactive IgG1 while the graft was functioning; however, we detected a strong alloantibody response on day 86 in animal E3. Co-staining with anti-CD20 demonstrated that alloantibodies were reacting against donor B cells (Figure 6C and D) suggesting specificity for MHC class II. ECDI treatment induces cell apoptosis A sample of ECDI-treated splenocytes was cultured for 5 days to assay apoptosis induced by ECDI treatment. Cells were stained for apoptosis markers annexin V and 7AAD or Live/Dead stain from Invitrogen (Woburn, MA). Untreated cells and irradiated cells served as controls for levels of apoptosis. Flow cytometry verified that a large American Journal of Transplantation 2015; 15: 2739–2749
proportion of cells were in the early stages of apoptosis immediately posttreatment, and the percentage of apoptosis significantly increased with time (Figure 6E). After 5 days in culture, nearly all ECDI-treated cells were apoptotic in contrast to untreated or irradiated cells.
Discussion Gaining durable transplant survival without ongoing immunosuppression has been held for decades as the prized goal of transplant research. For islet transplantation, in particular, a reliable and safe tolerance regimen could lead to broad expansion of the indication for islet transplant because of the delicate risk:benefit balance when considering the risks of transplant immunosuppression versus those of diabetes. In the current 2745
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Figure 6: Absence of alloantibody response upon ECDI-DLI treatment and ECDI-treated splenocytes undergo apoptosis. Serum samples were incubated with donor PBMCs followed by FITC-conjugated mouse anti-human IgG1 mAb and analyzed by flow cytometry. (A) Animal E4 posttransplant serum from POD 14 (early timepoint) and 64 (late timepoint) when animal still had excellent glycemic control demonstrated no alloantibody response. (B) Animal E3 at POD 36 (early timepoint) showed no detectable alloantibody response, but POD 86 (late timepoint) at time of rejection, demonstrated a strong alloantibody response. (C and D) Co-staining of E3 POD 86 (late timepoint) serum demonstrated that alloantibody-positive PBMCs were predominantly CD20þ cells. (E) Samples of NHPs splenocytes that were ECDI treated for 1 h were stained for with 7-AAD and annexin V. ECDI-treated cells were significantly more apoptotic than fresh or irradiated cells on day 0. After 5 days in culture, nearly all ECDI-treated cells were apoptotic in contrast to controls.
work, we explored immunosuppressive therapy designed to generate donor-specific Tregs in vivo in the recipient using Treg-promoting immunomodulatory agents. We found prolonged survival of NHP islet allografts after discontinuation of all immunosuppression at 4 weeks. 2746
Despite the positive nature of our findings, permanent survival and tolerance was not observed. Noteworthy is that our pilot study was designed to reveal a favorable immunomodulatory impact of ECDI-DLI rather than tolerance. We suspect that a more prolonged protocol of American Journal of Transplantation 2015; 15: 2739–2749
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maintenance immunosuppression with rapamycin, as used by Liu et al to 200 days (19) may better reveal the full tolerogenic potential of this approach. Additionally, it may be advantageous to provide a broader exposure to donor antigen by administering repeated doses as suggested by small animal studies (12,13). ECDI has been extensively studied as a means to reestablish tolerance to autoantigens by targeting T celldependent autoimmune responses seen in experimental autoimmune encephalomyelitis and autoimmune NOD diabetes (20,21). The encouraging success of these studies has led to early phase ECDI trials in relapsing multiple sclerosis (22,23). In addition to the clinical applicability of this approach, study of autoimmune models has yielded mechanistic understanding of ECDI-mediated tolerance. One key finding is that ECDI induces apoptosis in treated lymphocytes, likely explaining prompt clearance of ECDItreated cells postinfusion. Apoptotic cells target the indirect pathway via cross presentation of antigen by recipient APCs (24). Mechanistically, uptake of apoptotic cells is a component of a natural pathway of peripheral selftolerance (25–27). In the absence of danger signals associated with cell necrosis that would lead to DC activation and maturation, the ‘‘quiet’’ clearance of apoptotic cells leads to presentation of self-antigens by immature DCs that have tolerogenic propensity. This has been capitalized on in numerous experiments utilizing immature DCs as tolerogens (28), including ECDI treatment of DCs and feeding DCs apoptotic cells to maintain them in an immature-tolerogenic state (27). For studies targeting autoimmunity, presentation and tolerization via the indirect pathway is ideal, but to achieve the goal of transplant tolerance, both direct and indirect pathways need to be defeated; the fact that ECDI-treated APCs also renders directly responding T cells anergic makes the ECDI-based approach attractive for promoting transplant survival (11,29). Our rationale for the use of Thymoglobulin in NHP was to reduce effector cell number to generate a favorable Treg: Teffector ratio. In retrospect, the decision to employ a Thymo-based depleting induction regimen may have been misguided as Thymo is known to cause cytokine release and killing of targeted lymphocytes may be counter-productive by eliciting DC activation/maturation. In addition, although we separated the Thymo doses from islet and ECDI-DLI by 6 days, it is possible that there is residual Thymo in the circulation that binds the infused donor cells rerouting their clearance by different pathways that may not be tolerance promoting. Also potentially confounding is that T cell depletion induces homeostatic proliferation, a process that may impede tolerance (30). Studies to determine whether adjunctive nonlymphocytedepleting immunosuppression altered the timing of cell delivery are underway. In fact, in ongoing studies, a single animal receiving allogeneic islets has survival >190 days, after treatment with ECDI-DLI on d-3, d-0 with an adjunctive American Journal of Transplantation 2015; 15: 2739–2749
Treg promoting regimen of rapamycin, IL-6R blockade and a 10-day course of low-dose IL-2 (unpublished results). The studies we conducted are the first of which we are aware that seek to translate the promising small animal data using ECDI-DLI as a means to promote tolerance in a preclinical transplant model. Our findings suggest that infusion of donor ECDI-DLI at the time of allogeneic islet transplantation significantly prolongs allograft survival and is associated with Treg expansion, transient donor-specific hyporesponsiveness and absence of donor alloantibody production. Despite the positive nature of these findings, permanent survival and tolerance was not achieved. We continue our research to modify this regimen to attain the goal of durable tolerance.
Acknowledgments This work was supported in part by NIH grant: R00000000004607 to Emory University (ID: R00000000002176). Sub-award to Massachusetts General Hospital, J.F.M./D.H.S. (NIH 5U19A1051731-10) and NIH-NIDDK: 5K08DK094965-03 to H.Y. We also acknowledge the support of the NIAID/NIDDK Nonhuman Primate Transplantation Tolerance Cooperative Studies Group.
Authors’ Contributions J.F.M., J.L. designed research with the assistance of J.I.K., G.B., T.K., Y.Y., D.H.S.; J.L. conducted the islet isolation, transplantation, biopsy, drug administration, animal care, blood glucose and immune monitoring with the assistance of S.S., X.Z., Z.M., C.S., T.O., S.B., P.N.S.B., O.N., K.L.H.Y.; S.L., S.S., T.O., J.L. performed in vitro T and B cell flow cytometric and ELISPOT assays; R.S., J.I.K performed the mixed lymphocyte reaction assay and analysis; E.F., I.R., J.L. performed histology and immunohistochemical analysis; J.L., J.F.M. analyzed data and wrote the manuscript with the assistance of J.I.K. and C.S.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972; 72: 175–186. 2. Reckard CR, Ziegler MM, Barker CF. Physiological and immunological consequences of transplanting isolated pancreatic islets. Surgery 1973; 74: 91–99. 3. Fiorina P, Folli F, Bertuzzi F, et al. Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients. Diabetes care 2003; 26: 1129–1136.
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Lei et al 4. Warnock GL, Thompson DM, Meloche RM, et al. A multi-year analysis of islet transplantation compared with intensive medical therapy on progression of complications in type 1 diabetes. Transplantation 2008; 86: 1762–1766. 5. Thompson DM, Meloche M, Ao Z, et al. Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy. Transplantation 2011; 91: 373–378. 6. Bellin MD, Barton FB, Heitman A, et al. Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes. Am J Transplant 2012; 12: 1576–1583. 7. Turgeon NA, Avila JG, Cano JA, et al. Experience with a novel efalizumab-based immunosuppressive regimen to facilitate single donor islet cell transplantation. Am J Transplant 2010; 10: 2082– 2091. 8. Shapiro AM, Geng Hao E, Lakey JR, Finegood DT, Rajotte RV, Kneteman NM. Defining optimal immunosuppression for islet transplantation based on reduced diabetogenicity in canine islet autografts. Transplantation 2002; 74: 1522–1528. 9. Hyder A, Laue C, Schrezenmeir J. Effect of the immunosuppressive regime of Edmonton protocol on the long-term in vitro insulin secretion from islets of two different species and age categories. Toxicol In Vitro 2005; 19: 541–546. 10. Opelz G, Graver B, Terasaki PI. Induction of high kidney graft survival rate by multiple transfusion. Lancet 1981; 1: 1223–1225. 11. Jenkins MK, Schwartz RH. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 1987; 165: 302–319. 12. Chen G, Kheradmand T, Bryant J, et al. Intragraft CD11b(þ) IDO(þ) cells mediate cardiac allograft tolerance by ECDI-fixed donor splenocyte infusions. Am J Transplant 2012; 12: 2920–2929. 13. Luo X, Pothoven KL, McCarthy D, et al. ECDI-fixed allogeneic splenocytes induce donor-specific tolerance for long-term survival of islet transplants via two distinct mechanisms. Proc Natl Acad Sci USA 2008; 105: 14527–14532. 14. Mihara M, Kotoh M, Nishimoto N, et al. Humanized antibody to human interleukin-6 receptor inhibits the development of collagen arthritis in cynomolgus monkeys. Clin Immunol 2001; 98: 319–326. 15. Ansite ANB J, Barbaro B, Battle J, et al. Purified human pancreatic islets (PHPI) master production batch record? A standard operating procedure of the NIH clinical islet transplantation consortium. CellR4 2014; 2: 891 16. Miller SD, Wetzig RP, Claman HN. The induction of cell-mediated immunity and tolerance with protein antigens coupled to syngeneic lymphoid cells. J Exp Med 1979; 149: 758–773. 17. Koulmanda M, Smith RN, Qipo A, Weir G, Auchincloss H, Strom TB. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure? Am J Transplant 2006; 6: 687–696. 18. Li Y, Zhao X, Cheng D, et al. The presence of Foxp3 expressing T cells within grafts of tolerant human liver transplant recipients. Transplantation 2008; 86: 1837–1843. 19. Liu C, Noorchashm H, Sutter JA, et al. B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates. Nat Med 2007; 13: 1295–1298. 20. Fife BT, Guleria I, Gubbels Bupp M, et al. Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway. J Exp Med 2006; 203: 2737–2747. 21. Prasad S, Kohm AP, McMahon JS, Luo X, Miller SD. Pathogenesis of NOD diabetes is initiated by reactivity to the insulin B chain 9–23 epitope and involves functional epitope spreading. J Autoimmun 2012; 39: 347–353.
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22. Turley DM, Miller SD. Prospects for antigen-specific tolerance based therapies for the treatment of multiple sclerosis. Results Probl Cell Differ 2010; 51: 217–235. 23. Miller SD, Turley DM, Podojil JR. Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat Rev Immunol 2007; 7: 665–677. 24. Kaneko K, Morelli AE, Wang Z, Thomson AW. Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival. Clin Immunol 2003; 108: 190–198. 25. Ferguson TA, Choi J, Green DR. Armed response: How dying cells influence T-cell functions. Immunol Rev 2011; 241: 77–88. 26. Morelli AE, Larregina AT. Apoptotic cell-based therapies against transplant rejection: Role of recipient’s dendritic cells. Apoptosis 2010; 15: 1083–1097. 27. Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells. Am J Transplant 2006; 6: 1297–1311. 28. Raimondi G, Thomson AW. Dendritic cells,tolerance and therapy of organ allograft rejection. Contrib Nephrol 2005; 146: 105–120. 29. Kheradmand T, Wang S, Bryant J, et al. Ethylenecarbodiimidefixed donor splenocyte infusions differentially target direct and indirect pathways of allorecognition for induction of transplant tolerance. J Immunol 2012; 189: 804–812. 30. Wu Z, Bensinger SJ, Zhang J, et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat Med 2004; 10: 87–92.
Supporting Information Additional Supporting Information may be found in the online version of this article. Supplemental Materials and Methods Figure S1: MHC disparity information. (A) Example of two pairs animals that are MHC fully mis-matched (both Class I and Class II). (B) Summarized MHC mismatch information for all animals. In all cases except that of E2 the pairs are classified as fully mismatched at class I and class II. *: For E2 donor and recipient we consider the pair haplomismatched based on incomplete MHC genotyping. Each animal has one inconclusive haplotype. If the two haplotypes are assumed the identical, which is most unlikely, the available data support MHC haplo mismatching as a worst case scenario (greatest possible compatibility).
Figure S2: Post-STZ, pretransplant BG and insulin administration. Post-STZ administration, all animals (E1–E4 and C1–2) manifested robust diabetes pretransplant. Despite each animal received range 15–25 units per day of insulin administration to prevent aggressively postSTZ ketosis, all animals still experienced very poor glycemic control (A–F).
Figure S3: IVGTT demonstrated glucose homeostasis pre- and postislet transplant. IVGTTs performed 5–8 weeks posttransplant showed normal glucose homeostasis similar to that of pre-STZ for all four experimental group American Journal of Transplantation 2015; 15: 2739–2749
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animals. Animal E4 (sacrificed due to desquamating palm injury) demonstrated normal provocative glucose homeostasis similar to that of pre-STZ at time(day 81) of sacrifice (D). For animal E2 there was no pre-STZ data (B). For E3(C) there was no post-STZ IVGTT data because animal suffered ketoacidosis on the IVGTT procedure day. Control group recipients rejected too quickly for IVGTT to be performed.
Figure S4: Additional experimental group animal graft histology and immunochemistry. Animal E1 was biopsied at day 146 when BGs were high. Histology revealed disrupted islet architecture and intense lymphocytes infiltrating (A), with diminished insulin staining (A2), intensive CD8þ (B1), and CD4þ (B2) cells infiltrating islets consistent with rejection. However, there were only few to no CD4 (B2 brown)/Foxp3 (B2 blue) positive cells in and around islets were noted. Animal E2 upon sacrificed at day 62 posttransplant, necropsy revealed occasional preserved islets in the liver with diminished insulin staining(not shown), but there was a predominant pattern of islets with lymphocyte infiltration consistent with rejection (C1). The most striking finding was disseminated CMV infection evident in kidney (C2) and the pancreas (not shown). Animal E4 was sacrificed at day 81 when still had excellent glycemic control due to palm injury from unclear causes. Autopsy revealed numerous well-granulated islets with minimal to no infiltration of lymphocytes (D1) with strong insulin stain (D2).
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Figure S5: Additional control group animal graft histology and immunochemistry. Necropsy of hyperglycemic animal C1 revealed ruptured islet structures, with heavy lymphocytes infiltration(A1) consistent with rejection, diminished insulin staining (A2) at which point we observed intensive CD20þ (A3), CD8þ (A4) and CD4þ (A5) cells infiltrating the islets. There were only few to no CD4 (A5 brown)/Foxp3 (A5 blue) positive cells in and around islets were noted. Necropsy of hyperglycemic animal C2 revealed completely ruptured islet structures, with extremely heavy lymphocytes infiltration (B1) consistent with rejection, almost no insulin staining (B2) at which points we observed very intensive CD20þ (B3), CD8þ (B4), and CD4þ (B5) cells infiltrating the islets. There were no CD4 (B5 brown)/Foxp3 (B5 blue) positive cells in and around islets were noted.
Figure S6: Treg percent change and absolute number count. Despite the expected variability among animals, each of the ECDI-treated animals (E1–4) demonstrated an increase in Tregs percentage (% of CD4þCD25þ cells) compared to that of pretreatment (naive). Absolute number (number/mL) of Tregs of all experimental animals increased except E3 in which the absolute number remained relatively stable. Although the timing of these changes varied between animals, all manifested increases within the first 5 weeks post-ECDI-DLI. The control animals did not manifest consistent increases in either Treg percentage or absolute number.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13329
Brief Communication
Pilot Study Evaluating Regulatory T Cell–Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model J. Lei1,*, J. I. Kim1, S. Shi1, X. Zhang1, Z. Machaidze1, S. Lee1, C. Schuetz1, P. N. Martins1, T. Oura1, E. A. Farkash2, I. A. Rosales2, R. N. Smith2, R. Stott1, K. M. Lee1, J. Soohoo1, S. Boskovic1, K. Cappetta1, O. M. Nadazdin1, Y. Yamada1, H. Yeh1, T. Kawai1, D. H. Sachs1, G. Benichou1 and J. F. Markmann1,* 1
Center for Transplantation Science, Massachusetts General Hospital, Harvard Medical School, Boston, MA 2 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA Corresponding authors: James F. Markmann and Ji Lei, [email protected]; [email protected]
The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(30 -dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, antiinterleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donorspecific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.
diabetic complications (3–5). Islet transplantation results have improved with insulin independence rates approaching or equaling those seen in whole organ pancreas transplants at 5 years (6,7). However, current immunosuppressive agents are not only toxic to the recipient but are specifically toxic to beta cells and may induce peripheral insulin resistance (8,9). Because the risks of chronic immunosuppression may exceed the risks of diabetes-related complications for the majority of diabetics, islet transplantation is limited to those with severe labile disease with hypoglycemic unawareness. Thus, the full potential of isolated islet transplantation will not be realized until islet survival can be secured without chronic immunosuppression. A conceptually attractive path to donor-specific tolerance is through delivery of donor antigen in a tolerogenic manner (10). However, a potential pitfall of donor antigen exposure is recipient sensitization. A theoretical solution to this problem is found in classic experiments from Jenkins and Schwartz who determined that 1-ethyl-3-(30 -dimethylaminopropyl)-carbodiimide (ECDI) treatment of stimulators in vitro resulted in donor-specific anergy in responding T cells in mixed lymphocyte reaction-type assays (11). In this study, we examined the translational potential of administering ECDI-treated donor lymphocytes as a means to deliver nonstimulatory and nonsensitizing donor antigen in a tolerogenic manner, an approach that was recently reported to attain islet and heart graft tolerance in mice (12,13).
Materials and Methods
Abbreviations: DLI, donor splenocytes infusion; ECDI, ethylenecarbodiimide; NHP, nonhuman primate
Further information is available in Supplemental Materials and Methods.
Received 26 October 2014, revised 01 March 2015 and accepted for publication 20 March 2015
Animals and pairing selection Donors and recipients were paired on the basis of ABO blood group compatibility and MHC mismatching (Figure S1). In each case, one donor (weight range 5.0–9.2 kg) was used for one recipient (weight range 3.9–6.2 kg).
Introduction Treatment regimen
Isolated pancreatic islet transplantation offers minimally invasive restoration of the beta cell mass in type I diabetes (1,2) with potential for prevention and reversal of
All control and treated recipient animals received therapy consisting of (Figure 1A) the following: (1) thymoglobulin (anti-thymocyte globulin-rabbit, Genzyme, Cambridge, MA) I.V. 10 mg/kg on day 7 and day 6; (2) tocilizumab
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Figure 1: Significant prolongation of allogeneic islet graft survival by ECDI-treated donor splenocyte infusion. (A) Treatment plan. All cynomolgus macaques (nonhuman primates [NHPs]) were given thymoglobulin (thymo) and anti-IL6R. Daily rapamycin (rapa) was administrated for 1 month starting on day 3. Allogeneic islets were transplanted on day 0. ECDI-DLI was delivered via portal vein on the same day along with islet transplantation for experimental group only. (B) Glycemic control was maintained for 133, 48, 80, and >81 days for four ECDI-DLI experiment group NHPs (E1–E4), and 9, 18 days for two control group NHPs (C1, C2). Mean survival time of the ECDI-DLI group 85.5 days versus non-ECDI group 13.5 days, p ¼ 0.0177.
(anti-IL6R, Chugai Pharm, Tokyo, Japan [14]) I.V. at a dose of 10 mg/kg on days 7, 0, 7, 14, 21; (3) rapamycin (LC Laboratories, Woburn, MA) at a dose of 0.2 mg/Kg sc daily for total 1 month starting on day 3 with target blood trough level of 10–20 ng/mL; (4) ECDI-DLI was administered only to experimental group animals on day 0; and (5) allogeneic islet transplant was performed on day 0. Both islets and ECDI-DLI were infused via a branch of the portal vein. Daily low-dose insulin (2–4 units) was administrated for the first 30 days posttransplant to promote islet engraftment by allowing islet ‘‘rest’’ for both control and experimental group animals.
Diabetes induction and management NHPs received streptozotocin 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 180 mg/dL or nonfasting BG >250 mg/dL. Insulin (Humulin R, Lilly, Indianapolis, IN) and Lantus (Lilly) was administered by sliding scale to achieve BG 50% purity by Dithizone stain were combined for transplantation. Final islet preparations were enumerated by manual counting and sizing, and converting islet particle number (IPN) to islet equivalents (IEQ) based on a 150-mm diameter. Islets were then put into 15 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 ready for transplantation. Under general anesthesia, a small midline abdominal incision was performed to cannulate a branch of superior mesentery vein with an 18-gauge catheter
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Tregs Expansion to Protect Islet Graft Survival to access the portal system. The islet preparation was slowly infused by gravity over a period of approximately 10–15 min.
ECDI-treated cell preparation and infusion ECDI-treated donor lymphoid cells were delivered to experimental group recipients intraportally immediately after islet infusion. The protocol for ECDI treatment was previously described (16) and included harvest of spleen and lymph node, processing into single cell suspensions, lysis of RBC’s and incubation of lymphoid cells with ECDI (Merck KGaA [Novabiochem], Darmstadt, Germany) 75 mg/mL per 3.2 108 cells at 48C for 1 h with gentle shaking followed by washing and filtering to remove cell clumps. Prior to I.V. injection, samples of ECDI-treated lymphoid cells were stained for apoptosis marker 7-AAD and annexin V (both eBioscience, San Diego, CA). At each transplant, ECDI-treated cells from a single donor were infused in a volume of 30 mL.
Statistical analysis Data were analyzed using GraphPad Prism (version 5, GraphPad Software). Graft survival between experimental and control groups was compared using Log-rank (Mantel-Cox) Test and Gehan–Breslow–Wilcoxon statistics. Other differences between experimental group and control were analyzed using the Student’s unpaired t-test and F-test. p-Values less than 0.05 were considered statistically significant.
Results Significant prolongation of allogeneic islet graft survival by ECDI-treated donor lymphoid cell infusion A total of six diabetic NHP, four in the ECDI-DLI experimental group (referred to as E1–E4) and two in the non-ECDI-DLI control group (C1 and C2), were transplanted with single donor islets. Details of the characteristics of the recipients and transplants are summarized in Table 1. For the ECDI-DLI experimental group, recipients received between 4.5 108 and 3.75 109 ECDI-treated donor lymphoid cells via portal vein infusion. Prompt and excellent glycemic control was maintained for 133, 48, 80 and >81 days for the experiment group, E1–E4, respectively (mean survival time [MST] ¼ 85.5 days. Figure 1B). In contrast, the control group of NHP which received an identical regimen absent ECDI-DLI maintained glycemic control for 9 and 18 days (MST ¼ 13.5 days, Figure 1B) (ECDI-DLI group vs. non-ECDI-DLI group, p < 0.0177).
In animal E1, ideal BG control persisted for 133 days until loss of graft function (Figure 2A). An intravenous glucose tolerance test (IVGTT) performed at 5 weeks posttransplant showed normal glucose disposal similar to pre-STZ administration (Figure S3A). Liver biopsy performed at 146 days revealed most islets were infiltrated by lymphocytes, consistent with rejection (Figure S4A and B). Animal E2 glycemic control continued for 48 days posttransplant (Figure 2B). IVGTT performed at 5 weeks posttransplant showed a normal BG response (Figure S3B). Glycemic control partially deteriorated at 8 weeks. This recipient demonstrated elevated alkaline phosphatase and lipase (4 upper limit of normal, data not shown), persisting throughout the post-STZ and transplant course. Upon sacrifice at day 62 posttransplant, necropsy revealed occasional well-preserved, insulin-positive islets in the liver, but most islets were infiltrated by lymphocytes, consistent with rejection (Figure S4C1 and data not shown). The most striking finding was disseminated CMV infection evident in kidney (Figure S4C2) and the pancreas. Animal E3 maintained glycemic control posttransplant for 50 days (Figure 2C), then required low-dose (4–6 units) insulin to maintain acceptable nonfasting glucose level. Because of the imperfect graft function, a liver biopsy was performed at day 68. Histology revealed numerous wellgranulated, insulin-positive islets with minimal to no infiltrating lymphocytes (Figure 3A1) suggesting the absence of rejection. We suspect the imperfect glycemic control with eventual hyperglycemia represented marginal mass exhaustion which can occur in NHP isografts (17); however, the lack of lymphocytes infiltration on biopsy could also be due to sampling error. IVGTT performed at 40 and 70 days posttransplant showed normal glucose homeostasis comparable to the pre-STZ period (Figure S3C). Animal E4 achieved glycemic control (Figure 2D) but was sacrificed at day 81 due to palm injury from unclear causes. Autopsy revealed well-preserved islets with no indication of rejection (Figure S4D1 and D2). IVGTT performed at 64 and
Table 1: Summary of donors, recipients, transplanted islets and ECDI-treated donor spleen cells Experimental group Recipient ID Recipient body weight (RBW) (kg) Donor body weight (kg) Donor pancreas weight (g) Islet particle number Number of islet equivalent (IEQ) Pack cell volume (mL) Islet purity (%) Islet viability (%) IEQ/RBW kg Total ECDI-treated donor spleen cell# ECDI-treated donor spleen Cell#/Kg RBW
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4.2 9.0 8.0 153000 112900 0.4 60 98 26 881 1.05 109 2.50 108
3.84 7.5 10.6 146000 125600 0.3 80 98 32 708 4.5 108 1.17 108
5.1 7.0 8.5 113900 103200 0.4 70 99 20 235 1.5 109 2.94 108
6.2 9.2 10.0 132000 107400 0.4 75 95 17 323 3.75 109 6.05 108
4.3 5.0 8.5 121500 112000 0.4 80 99 26 047 N/A N/A
3.9 5.7 8.7 130500 119500 0.4 80 99 30 641 N/A N/A
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Survival Post-transplant(days) Figure 2: Glycemic control posttransplant. Four ECDI-DLI experimental group and two control group diabetic NHPs each promptly achieved normoglycemia postallogeneic islet transplant. Daily low-dose (2–4 units) insulin (INS) was administrated to all experimental and control group animals to help initial islet engrafting for the first 30 days posttransplant. Blood glucose (BG) measurements are nonfasting random daily tests.
81 days posttransplant showed normal glucose homeostasis comparable to the pre-STZ period (Figure S3D). Graft survival is associated with the presence more of CD4þ Foxp3þ T cell in and around grafts To characterize lymphocytic graft infiltration, we performed biopsy when grafts were functional. Biopsy histology of E3 (Figure 3A) revealed numerous well-granulated islets (A1) with strong insulin staining (A2), minimal to no infiltration of CD20þ (A3), CD8þ (A4), or CD4þ (A5) cells into islets. Lymphocytes remain at the periphery to form a cuff around the islets. In contrast, necropsy of hyperglycemic animal E3 revealed disrupted islet architecture (Figure 3B) with diminished insulin staining (B2) at which points we observed intensive CD20þ (B3), CD8þ (B4), and CD4þ (B5) cells infiltrating the islets. Most notably, during normoglycemia, biopsy of grafts revealed that the number of CD4 (A5 brown)/ Foxp3 (A5 blue) positive cells in and around islets was consistently higher than at the time of rejection similar to the 2742
findings in mouse models (B5 blue) (13,18). Biopsy performed on one additional experimental animal (E4, data not shown) at the time of normoglycemia revealed the similar histological findings as E3 during normoglycemia. Necropsy histology assessment of recipient E1 (Figure S4A and B), E2 (Figure S4C), and two control animals C1 and C2 (Figure S5) was also performed when grafts were rejected. The pattern of islet destruction and cell infiltration was consistent among all rejected animals, experiential and control. Significant CD4þCD25þFoxp3þ generation and expansion associated with graft survival post-ECDI-DLI To study the dynamics of Treg levels in the circulation, PBMCs were stained for CD4, CD25, and Foxp3 at pretreatment (naive) and several time points post-ECDI-DLI. We observed a significant relative expansion in the proportion of CD4þCD25þFoxp3þ Tregs in PBMC by flow in all four ECDI-DLI recipients. The peak fold increase ranged from 9- to 21-fold compared to that of pretreatment levels (Figure 4A). American Journal of Transplantation 2015; 15: 2739–2749
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Figure 3: Islet graft histology and immunochemistry. Biopsy and necropsy graft samples were collected and sections were stained with H&E, insulin (brown), CD20, CD8, and CD4 (brown)/Foxp3 (blue). (A) At time of normoglycemia at day 68, biopsy of recipient E3 revealed well-granulated islets (A1) with strong insulin staining (A2), minimal to no infiltration of CD20þ (A3), CD8þ (A4), or CD4þ (A5) cells into islets. (B) Necropsy of grafts taken from hyperglycemia animals revealed ruptured islet structures (B1) with much diminished insulin stain (B2). Intense lymphocytic infiltration into the islets (B3, B4, B5) were observed. Most notably, biopsy of survival grafts revealed that the number of CD4 (brown)/Foxp3 (blue) positive cells (A5) in and around islets are higher compared with rejected grafts (B5). Photomicrographs are representative of five sections of a recipient (E3). The patterns of cell infiltration were consistent across animals studied. Arrows indicate islets. More detailed histology data at Figures S4 and S5.
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The detected Treg average peak of experimental group is significantly higher than that of control group (Figure 4B, p ¼ 0.0256). Peak expansion was detected during the first 1–5 weeks post-ECDI-DLI (Figure 4A). The detailed Treg dynamics of animal E3 (Figure 4C and D) is presented showing that Tregs peaked around day 5 post-ECDI-DLI and Treg levels remained elevated for about 3 weeks at which point levels gradually decreased but stabilized at levels higher than levels pretreatment. Similar trend was observed in all ECDI-DLI recipients. In the control group, neither recipient demonstrated an elevated percentage of Tregs suggesting a critical role of ECDI-DLI in the expansion of Tregs (detailed Treg percentage change and absolute Treg number count for all animals are presented in Figure S6). Lymphocytes from ECDI-DLI recipients exhibit donorspecific hyporesponsiveness To assess for donor-specific hyporesponsiveness, we examined alloreactivity of recipient PBMC by ELISPOT to
donor PBMC and to third party PBMC at multiple time points posttransplant. While normoglycemic (NG), all ECDIDLI recipients demonstrated significantly reduced responses to donor PBMC relative to third party PBMC as measured by IFN-g ELISPOT (Figure 5A–D). Donor-specific hyporesponsiveness was lost when assessed at the time of graft rejection (hyperglycemia, HG). In contrast, lymphocytes from recipients in the control group exhibited no donor-specific hyporesponsiveness during the period of normoglycemia (Figure 5E and F). These data suggest that donor-specific T effectors were rendered at least transiently unresponsive by the ECDI-DLI protocol. Absence of alloantibody response upon ECDI-DLI treatment We examined the development of anti-donor antibody (IgG1 isotype) in two ECDI-DLI recipients by flow cytometry. Animal E4 posttransplant serum samples on days 14 and 64, time points while the graft was still functioning, were analyzed. Flow
Figure 4: Significant Treg expansion in ECDI-DLI recipients. (A) All four ECDI-DLI experimental group diabetic NHPs but not control group demonstrated evidence of Treg expansion in PBMC by flow. The detected peak increase ranged from 9- to 21-fold compared to that of pretreatment (naive). The day of detected peak relative to transplant is shown on the top of each bar graph. (B) The detected average of fold increase of Treg of the experimental group is significantly higher than that of control group, p ¼ 0.0256. (C and D) Detailed flow of Treg levels of recipient E3 are shown from multiple time points. (C) High Treg levels were maintained for about 3 weeks before gradually decreasing in animal E3. More detailed histology data at Figure S6.
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NG: Normoglycemia HG: Hyperglycemia NA: Data not available Pre-Tx: Pre-treatment
Figure 5: ECDI-DLI induces donor-specific hyporesponsiveness. Responsiveness was assayed by measuring IFN-g production by ELISpot and recipient PBMCs were used as responders. (A–D) All four ECDI-DLI experimental group diabetic NHPs demonstrated evidence of donor-specific hyporesponsiveness during normoglycemia. Recipients PBMCs react remarkably less to donor cells than to third party demonstrated when grafts were functioning. Donor-specific hyporesponsiveness was lost when grafts were rejected. For animal E1 and E2 (A and B), there are no pretreatment (naive) data available. (E and F) Control recipients without ECDI-DLI showed no donor-specific hyporesponsiveness during normoglycemia period of study.
cytometry revealed no alloantibody response at these two time points (Figure 6A). Animal E3 posttransplant serum samples on days 36 and day 86 posttransplant, before and after rejection, were analyzed (Figure 6B). As with E4, E3 exhibited no donorreactive IgG1 while the graft was functioning; however, we detected a strong alloantibody response on day 86 in animal E3. Co-staining with anti-CD20 demonstrated that alloantibodies were reacting against donor B cells (Figure 6C and D) suggesting specificity for MHC class II. ECDI treatment induces cell apoptosis A sample of ECDI-treated splenocytes was cultured for 5 days to assay apoptosis induced by ECDI treatment. Cells were stained for apoptosis markers annexin V and 7AAD or Live/Dead stain from Invitrogen (Woburn, MA). Untreated cells and irradiated cells served as controls for levels of apoptosis. Flow cytometry verified that a large American Journal of Transplantation 2015; 15: 2739–2749
proportion of cells were in the early stages of apoptosis immediately posttreatment, and the percentage of apoptosis significantly increased with time (Figure 6E). After 5 days in culture, nearly all ECDI-treated cells were apoptotic in contrast to untreated or irradiated cells.
Discussion Gaining durable transplant survival without ongoing immunosuppression has been held for decades as the prized goal of transplant research. For islet transplantation, in particular, a reliable and safe tolerance regimen could lead to broad expansion of the indication for islet transplant because of the delicate risk:benefit balance when considering the risks of transplant immunosuppression versus those of diabetes. In the current 2745
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Figure 6: Absence of alloantibody response upon ECDI-DLI treatment and ECDI-treated splenocytes undergo apoptosis. Serum samples were incubated with donor PBMCs followed by FITC-conjugated mouse anti-human IgG1 mAb and analyzed by flow cytometry. (A) Animal E4 posttransplant serum from POD 14 (early timepoint) and 64 (late timepoint) when animal still had excellent glycemic control demonstrated no alloantibody response. (B) Animal E3 at POD 36 (early timepoint) showed no detectable alloantibody response, but POD 86 (late timepoint) at time of rejection, demonstrated a strong alloantibody response. (C and D) Co-staining of E3 POD 86 (late timepoint) serum demonstrated that alloantibody-positive PBMCs were predominantly CD20þ cells. (E) Samples of NHPs splenocytes that were ECDI treated for 1 h were stained for with 7-AAD and annexin V. ECDI-treated cells were significantly more apoptotic than fresh or irradiated cells on day 0. After 5 days in culture, nearly all ECDI-treated cells were apoptotic in contrast to controls.
work, we explored immunosuppressive therapy designed to generate donor-specific Tregs in vivo in the recipient using Treg-promoting immunomodulatory agents. We found prolonged survival of NHP islet allografts after discontinuation of all immunosuppression at 4 weeks. 2746
Despite the positive nature of our findings, permanent survival and tolerance was not observed. Noteworthy is that our pilot study was designed to reveal a favorable immunomodulatory impact of ECDI-DLI rather than tolerance. We suspect that a more prolonged protocol of American Journal of Transplantation 2015; 15: 2739–2749
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maintenance immunosuppression with rapamycin, as used by Liu et al to 200 days (19) may better reveal the full tolerogenic potential of this approach. Additionally, it may be advantageous to provide a broader exposure to donor antigen by administering repeated doses as suggested by small animal studies (12,13). ECDI has been extensively studied as a means to reestablish tolerance to autoantigens by targeting T celldependent autoimmune responses seen in experimental autoimmune encephalomyelitis and autoimmune NOD diabetes (20,21). The encouraging success of these studies has led to early phase ECDI trials in relapsing multiple sclerosis (22,23). In addition to the clinical applicability of this approach, study of autoimmune models has yielded mechanistic understanding of ECDI-mediated tolerance. One key finding is that ECDI induces apoptosis in treated lymphocytes, likely explaining prompt clearance of ECDItreated cells postinfusion. Apoptotic cells target the indirect pathway via cross presentation of antigen by recipient APCs (24). Mechanistically, uptake of apoptotic cells is a component of a natural pathway of peripheral selftolerance (25–27). In the absence of danger signals associated with cell necrosis that would lead to DC activation and maturation, the ‘‘quiet’’ clearance of apoptotic cells leads to presentation of self-antigens by immature DCs that have tolerogenic propensity. This has been capitalized on in numerous experiments utilizing immature DCs as tolerogens (28), including ECDI treatment of DCs and feeding DCs apoptotic cells to maintain them in an immature-tolerogenic state (27). For studies targeting autoimmunity, presentation and tolerization via the indirect pathway is ideal, but to achieve the goal of transplant tolerance, both direct and indirect pathways need to be defeated; the fact that ECDI-treated APCs also renders directly responding T cells anergic makes the ECDI-based approach attractive for promoting transplant survival (11,29). Our rationale for the use of Thymoglobulin in NHP was to reduce effector cell number to generate a favorable Treg: Teffector ratio. In retrospect, the decision to employ a Thymo-based depleting induction regimen may have been misguided as Thymo is known to cause cytokine release and killing of targeted lymphocytes may be counter-productive by eliciting DC activation/maturation. In addition, although we separated the Thymo doses from islet and ECDI-DLI by 6 days, it is possible that there is residual Thymo in the circulation that binds the infused donor cells rerouting their clearance by different pathways that may not be tolerance promoting. Also potentially confounding is that T cell depletion induces homeostatic proliferation, a process that may impede tolerance (30). Studies to determine whether adjunctive nonlymphocytedepleting immunosuppression altered the timing of cell delivery are underway. In fact, in ongoing studies, a single animal receiving allogeneic islets has survival >190 days, after treatment with ECDI-DLI on d-3, d-0 with an adjunctive American Journal of Transplantation 2015; 15: 2739–2749
Treg promoting regimen of rapamycin, IL-6R blockade and a 10-day course of low-dose IL-2 (unpublished results). The studies we conducted are the first of which we are aware that seek to translate the promising small animal data using ECDI-DLI as a means to promote tolerance in a preclinical transplant model. Our findings suggest that infusion of donor ECDI-DLI at the time of allogeneic islet transplantation significantly prolongs allograft survival and is associated with Treg expansion, transient donor-specific hyporesponsiveness and absence of donor alloantibody production. Despite the positive nature of these findings, permanent survival and tolerance was not achieved. We continue our research to modify this regimen to attain the goal of durable tolerance.
Acknowledgments This work was supported in part by NIH grant: R00000000004607 to Emory University (ID: R00000000002176). Sub-award to Massachusetts General Hospital, J.F.M./D.H.S. (NIH 5U19A1051731-10) and NIH-NIDDK: 5K08DK094965-03 to H.Y. We also acknowledge the support of the NIAID/NIDDK Nonhuman Primate Transplantation Tolerance Cooperative Studies Group.
Authors’ Contributions J.F.M., J.L. designed research with the assistance of J.I.K., G.B., T.K., Y.Y., D.H.S.; J.L. conducted the islet isolation, transplantation, biopsy, drug administration, animal care, blood glucose and immune monitoring with the assistance of S.S., X.Z., Z.M., C.S., T.O., S.B., P.N.S.B., O.N., K.L.H.Y.; S.L., S.S., T.O., J.L. performed in vitro T and B cell flow cytometric and ELISPOT assays; R.S., J.I.K performed the mixed lymphocyte reaction assay and analysis; E.F., I.R., J.L. performed histology and immunohistochemical analysis; J.L., J.F.M. analyzed data and wrote the manuscript with the assistance of J.I.K. and C.S.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972; 72: 175–186. 2. Reckard CR, Ziegler MM, Barker CF. Physiological and immunological consequences of transplanting isolated pancreatic islets. Surgery 1973; 74: 91–99. 3. Fiorina P, Folli F, Bertuzzi F, et al. Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients. Diabetes care 2003; 26: 1129–1136.
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Lei et al 4. Warnock GL, Thompson DM, Meloche RM, et al. A multi-year analysis of islet transplantation compared with intensive medical therapy on progression of complications in type 1 diabetes. Transplantation 2008; 86: 1762–1766. 5. Thompson DM, Meloche M, Ao Z, et al. Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy. Transplantation 2011; 91: 373–378. 6. Bellin MD, Barton FB, Heitman A, et al. Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes. Am J Transplant 2012; 12: 1576–1583. 7. Turgeon NA, Avila JG, Cano JA, et al. Experience with a novel efalizumab-based immunosuppressive regimen to facilitate single donor islet cell transplantation. Am J Transplant 2010; 10: 2082– 2091. 8. Shapiro AM, Geng Hao E, Lakey JR, Finegood DT, Rajotte RV, Kneteman NM. Defining optimal immunosuppression for islet transplantation based on reduced diabetogenicity in canine islet autografts. Transplantation 2002; 74: 1522–1528. 9. Hyder A, Laue C, Schrezenmeir J. Effect of the immunosuppressive regime of Edmonton protocol on the long-term in vitro insulin secretion from islets of two different species and age categories. Toxicol In Vitro 2005; 19: 541–546. 10. Opelz G, Graver B, Terasaki PI. Induction of high kidney graft survival rate by multiple transfusion. Lancet 1981; 1: 1223–1225. 11. Jenkins MK, Schwartz RH. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 1987; 165: 302–319. 12. Chen G, Kheradmand T, Bryant J, et al. Intragraft CD11b(þ) IDO(þ) cells mediate cardiac allograft tolerance by ECDI-fixed donor splenocyte infusions. Am J Transplant 2012; 12: 2920–2929. 13. Luo X, Pothoven KL, McCarthy D, et al. ECDI-fixed allogeneic splenocytes induce donor-specific tolerance for long-term survival of islet transplants via two distinct mechanisms. Proc Natl Acad Sci USA 2008; 105: 14527–14532. 14. Mihara M, Kotoh M, Nishimoto N, et al. Humanized antibody to human interleukin-6 receptor inhibits the development of collagen arthritis in cynomolgus monkeys. Clin Immunol 2001; 98: 319–326. 15. Ansite ANB J, Barbaro B, Battle J, et al. Purified human pancreatic islets (PHPI) master production batch record? A standard operating procedure of the NIH clinical islet transplantation consortium. CellR4 2014; 2: 891 16. Miller SD, Wetzig RP, Claman HN. The induction of cell-mediated immunity and tolerance with protein antigens coupled to syngeneic lymphoid cells. J Exp Med 1979; 149: 758–773. 17. Koulmanda M, Smith RN, Qipo A, Weir G, Auchincloss H, Strom TB. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure? Am J Transplant 2006; 6: 687–696. 18. Li Y, Zhao X, Cheng D, et al. The presence of Foxp3 expressing T cells within grafts of tolerant human liver transplant recipients. Transplantation 2008; 86: 1837–1843. 19. Liu C, Noorchashm H, Sutter JA, et al. B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates. Nat Med 2007; 13: 1295–1298. 20. Fife BT, Guleria I, Gubbels Bupp M, et al. Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway. J Exp Med 2006; 203: 2737–2747. 21. Prasad S, Kohm AP, McMahon JS, Luo X, Miller SD. Pathogenesis of NOD diabetes is initiated by reactivity to the insulin B chain 9–23 epitope and involves functional epitope spreading. J Autoimmun 2012; 39: 347–353.
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22. Turley DM, Miller SD. Prospects for antigen-specific tolerance based therapies for the treatment of multiple sclerosis. Results Probl Cell Differ 2010; 51: 217–235. 23. Miller SD, Turley DM, Podojil JR. Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat Rev Immunol 2007; 7: 665–677. 24. Kaneko K, Morelli AE, Wang Z, Thomson AW. Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival. Clin Immunol 2003; 108: 190–198. 25. Ferguson TA, Choi J, Green DR. Armed response: How dying cells influence T-cell functions. Immunol Rev 2011; 241: 77–88. 26. Morelli AE, Larregina AT. Apoptotic cell-based therapies against transplant rejection: Role of recipient’s dendritic cells. Apoptosis 2010; 15: 1083–1097. 27. Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells. Am J Transplant 2006; 6: 1297–1311. 28. Raimondi G, Thomson AW. Dendritic cells,tolerance and therapy of organ allograft rejection. Contrib Nephrol 2005; 146: 105–120. 29. Kheradmand T, Wang S, Bryant J, et al. Ethylenecarbodiimidefixed donor splenocyte infusions differentially target direct and indirect pathways of allorecognition for induction of transplant tolerance. J Immunol 2012; 189: 804–812. 30. Wu Z, Bensinger SJ, Zhang J, et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat Med 2004; 10: 87–92.
Supporting Information Additional Supporting Information may be found in the online version of this article. Supplemental Materials and Methods Figure S1: MHC disparity information. (A) Example of two pairs animals that are MHC fully mis-matched (both Class I and Class II). (B) Summarized MHC mismatch information for all animals. In all cases except that of E2 the pairs are classified as fully mismatched at class I and class II. *: For E2 donor and recipient we consider the pair haplomismatched based on incomplete MHC genotyping. Each animal has one inconclusive haplotype. If the two haplotypes are assumed the identical, which is most unlikely, the available data support MHC haplo mismatching as a worst case scenario (greatest possible compatibility).
Figure S2: Post-STZ, pretransplant BG and insulin administration. Post-STZ administration, all animals (E1–E4 and C1–2) manifested robust diabetes pretransplant. Despite each animal received range 15–25 units per day of insulin administration to prevent aggressively postSTZ ketosis, all animals still experienced very poor glycemic control (A–F).
Figure S3: IVGTT demonstrated glucose homeostasis pre- and postislet transplant. IVGTTs performed 5–8 weeks posttransplant showed normal glucose homeostasis similar to that of pre-STZ for all four experimental group American Journal of Transplantation 2015; 15: 2739–2749
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animals. Animal E4 (sacrificed due to desquamating palm injury) demonstrated normal provocative glucose homeostasis similar to that of pre-STZ at time(day 81) of sacrifice (D). For animal E2 there was no pre-STZ data (B). For E3(C) there was no post-STZ IVGTT data because animal suffered ketoacidosis on the IVGTT procedure day. Control group recipients rejected too quickly for IVGTT to be performed.
Figure S4: Additional experimental group animal graft histology and immunochemistry. Animal E1 was biopsied at day 146 when BGs were high. Histology revealed disrupted islet architecture and intense lymphocytes infiltrating (A), with diminished insulin staining (A2), intensive CD8þ (B1), and CD4þ (B2) cells infiltrating islets consistent with rejection. However, there were only few to no CD4 (B2 brown)/Foxp3 (B2 blue) positive cells in and around islets were noted. Animal E2 upon sacrificed at day 62 posttransplant, necropsy revealed occasional preserved islets in the liver with diminished insulin staining(not shown), but there was a predominant pattern of islets with lymphocyte infiltration consistent with rejection (C1). The most striking finding was disseminated CMV infection evident in kidney (C2) and the pancreas (not shown). Animal E4 was sacrificed at day 81 when still had excellent glycemic control due to palm injury from unclear causes. Autopsy revealed numerous well-granulated islets with minimal to no infiltration of lymphocytes (D1) with strong insulin stain (D2).
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Figure S5: Additional control group animal graft histology and immunochemistry. Necropsy of hyperglycemic animal C1 revealed ruptured islet structures, with heavy lymphocytes infiltration(A1) consistent with rejection, diminished insulin staining (A2) at which point we observed intensive CD20þ (A3), CD8þ (A4) and CD4þ (A5) cells infiltrating the islets. There were only few to no CD4 (A5 brown)/Foxp3 (A5 blue) positive cells in and around islets were noted. Necropsy of hyperglycemic animal C2 revealed completely ruptured islet structures, with extremely heavy lymphocytes infiltration (B1) consistent with rejection, almost no insulin staining (B2) at which points we observed very intensive CD20þ (B3), CD8þ (B4), and CD4þ (B5) cells infiltrating the islets. There were no CD4 (B5 brown)/Foxp3 (B5 blue) positive cells in and around islets were noted.
Figure S6: Treg percent change and absolute number count. Despite the expected variability among animals, each of the ECDI-treated animals (E1–4) demonstrated an increase in Tregs percentage (% of CD4þCD25þ cells) compared to that of pretreatment (naive). Absolute number (number/mL) of Tregs of all experimental animals increased except E3 in which the absolute number remained relatively stable. Although the timing of these changes varied between animals, all manifested increases within the first 5 weeks post-ECDI-DLI. The control animals did not manifest consistent increases in either Treg percentage or absolute number.
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