Recurrence of type 1 diabetes following simultaneous pancreas-kidney transplantation

C H A P T E R

24 Recurrence of type 1 diabetes following simultaneous pancreas-kidney transplantation George W. Burke, III⁎,†,‡, Gaetano Ciancio⁎,†, Mahmoud Morsi⁎,†, Jose Figueiro⁎,†, Linda Chen⁎,†, Francesco Vendrame§, Alberto Pugliese‡,§,¶ ⁎

Miami Transplant Institute, Jackson Memorial Hospital, University of Miami Miller School of Medicine, Miami, FL, United States †Department of Surgery, Division of Transplantation, University of Miami Miller School of Medicine, Miami, FL, United States ‡Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States §Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Miami Miller School of Medicine, Miami, FL, United States ¶Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, United States

O U T L I N E Preamble

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Introduction 297 Diagnosis of T1D recurrence (T1DR) 297 Longitudinal study of T1DR at the University of Miami 304 Therapy for T1D 306 T1DR therapy at the Miami Transplant Institute 307 Theoretical considerations 307 Combination therapies 308

Preamble The University of Miami Miller School of Medicine/Miami Transplant Institute simultaneous ­pancreas-kidney (SPK) transplant program began in June 1990. Nearly, all of the >600 SPK recipients have been diagnosed with type 1 diabetes (T1D) and endstage renal disease (ESRD). Since then our experience has been an odyssey including an evolution in the approach to SPK recipient coagulation status, surgical procedure, immunosuppression, patient care, and research involving pancreas transplant outcome and an observation that autoimmunity can recur in the pancreas transplant years later.

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 1 https://doi.org/10.1016/B978-0-12-814833-4.00024-1

Future considerations

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Network for the pancreas organ donors with diabetes

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Conclusions

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Acknowledgment

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References

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Our odyssey provided a number of observations along the way. One of the early findings was the identification of a hypercoagulable state associated with kidney-pancreas transplantation, specifically in individuals with long-standing T1D and ESRD. This was supported by data obtained from an intraoperative thromboelastogram (TEG) during SPK surgery.1 This was significant at the time, since there was a sense that the uremic state of patients with T1D/ESRD would have an effect on platelets, rendering patients more likely to bleed after transplant surgery. This finding lead to the judicious use of anticoagulation, heparin, or other agents, in an attempt to minimize the likelihood of venous thrombosis following pancreas transplantation. Furthermore this ­provided

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substantiation for the application of the concept of Virchow’s triad to pancreas transplant thrombosis as an explanation for why the pancreas is so much more prone to thrombosis than other solid organs. Virchow’s triad to explain thrombosis incorporates: (1) endothelial damage, (2) hypercoagulability, and (3) venous stasis. All solid organ transplants experience some element of endothelial damage related to the ischemia/reperfusion injury. Hypercoagulability in our patient population was demonstrated by TEG as well as laboratory data including elevated fibrinogen levels, prothrombin time, international normalized ratio, partial thromboplastin time, and platelet count.1 Venous stasis, specifically related to the pancreas transplant, develops once the large veins including superior mesenteric vein, and splenic vein are ligated, removing small intestines and spleen, respectively, as sources of blood return through both large veins. This leads to an obligatory state of venous stasis in the pancreas transplant. Thus, Virchow’s triad provides a compelling explanation for the higher incidence of venous thrombosis in pancreas transplants compared to other solid organ transplants.1 The surgical approach to both the retrieval of the pancreas allograft,2 as well as the backtable preparation of the pancreas for transplantation,3 has been carefully developed over the years, to optimize outcome. In our series, the donor duodenum size has been kept as small as possible, to minimize potential complications related to bladder drainage of the pancreas transplant. Another feature important in the development of surgical techniques to improve pancreas transplant outcome included the use of arterial reconstruction with donor iliac arteries during the pancreas transplant procedure as an intraoperative approach to arterial injury or inadequate flow within native iliac arteries due to severe diabetes-­ atherosclerosis-related narrowing.4 This option allowed for the replacement of a severely narrowed, atherosclerotic recipient external iliac artery with an essentially normal piece of donor external iliac artery,4,5 improving flow both to the ipsilateral lower extremity, and to the kidney and/or pancreas transplant. Immunosuppression, including both induction and maintenance therapy, also evolved significantly since the start of the pancreas transplant program. Thymoglobulin, which has been our standard induction therapy since 1998, includes a number of membrane surface targets such as CD3/T-cell receptor (TCR), CD4, CD8, HLA class I and II, costimulatory molecules including B7-1 and B7-2, as well as CD28 and CTLLA-4, chemokine receptors including CXCR4, as well as CD2, CD40,6 and B cell markers.7 In addition, thymoglobulin was shown to provide protection from the ischemia/reperfusion injury.8 Although thymoglobulin was noted to target a panoply of important immunological receptors it did not appear to contain significant activity against the IL-2

receptor (CD25), which is a marker of T cell activation. For this reason, monoclonal antibody to IL-2 receptor was added to thymoglobulin as part of our induction protocol. This resulted in the minimization of peripheral blood CD25+ T cells for 30 days and up to 45 days following transplantation.9 Our experience with this combined induction therapy led to the initiation of a randomized, prospective immunosuppressive trial using thymoglobulin and anti-CD25 monoclonal antibody induction therapy as well as tacrolimus and steroid maintenance therapy, with randomization to rapamycin (Rapa) or mycophenolate mofetil (MMF) for SPK transplants.10 This study enrolled 170 patients of whom 84 were in the Rapa arm, and 86 in the MMF arm. Several unique features of the University of Miami SPK program include: (1) c-peptide confirmation of T1D, (2) bladder drainage of the exocrine pancreas,10,11 (3) stored sera for most patients, (4) a high percentage of minorities with T1D, and (5) prolonged cold ischemia time (CIT), with a mean of 20–22 h of CIT in both arms. Approximately 25% of patients enrolled in this study had a history of coronary artery disease. Results at 10 years included statistically significantly lower rates of biopsy-proven acute rejection for both kidney and pancreas transplants with rapamycin when compared with MMF. However, there was no difference between the two groups for creatinine or c-peptide at 10  years. Furthermore, there was no difference in patient, pancreas transplant or kidney transplant survival at 10 years. Most of the deaths were related to cardiovascular disease. Notably, the pancreas transplant survival at 10 years (death-censored) was 98% in the Rapa and 84% in the MMF arm although the differences were not statistically significant (Fig. 1A). Kidney transplant survival at 10 years (death-censored) was 74% in the Rapa and 70% in the MMF arm (Fig. 1B). Our enteric conversion rate was approximately 10%. Ten-year patient survival was approximately 70%; overall 10-year pancreas graft survival was 91% (death-censored); and overall 10-year kidney graft survival was 72% (death-­ censored).10 Other results included a low incidence of viral infections, lymphoproliferative disorders, and acute rejection.10,12 This experience leads to our ongoing studies of SPK recipients and long-term outcome. An important observation was that a small number of patients presented several years after transplantation with severe hyperglycemia and ketoacidosis in the context of low to absent initial c-peptide levels, yet no change in exocrine pancreas transplant (urine amylase) or kidney transplant (serum creatinine) function. This observation suggested that the sudden onset of hyperglycemia was possibly caused by a specific insult at the level of the pancreas islet beta cell.

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FIG.  1  SPKT recipients treated with rapamycin vs MMF: (A) % pancreas death-censored survival and (B) % kidney death-censored graft survival. Reproduced from Ciancio G, Sageshima J, Chen L, Gaynor JJ, Hanson L, Tueros L, Montenora-Velards E, Gomez C, Kupin W, Guerra G, Mattiazzi A, Fornoni A, Pugliese A, Roth D, Wolf M, Burke GW III. Advantage of rapamycin over mycophenolate mofetil when used with tacrolimus for simultaneous pancreas kidney transplants: randomized, single-­ center trial at ten years. Am J Transplant 2012; 12(12):3363–3376. Copyright 1999–2018 John Wiley and Sons, Inc. All rights reserved.

This lead to the study of recurrence of autoimmunity in SPK recipients at the University of Miami/Miami Transplant Institute.

Introduction Diagnosis of T1D recurrence (T1DR) Type 1 diabetes recurrence (T1DR) following pancreas transplantation was first described by David ER Sutherland in HLA identical twins or sibling pairs who, therefore, received no or minimal immunosuppression, in the 1980s.13 For SPK recipients, immunosuppression was used routinely, and it was felt that

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i­mmunosuppression that was sufficient to prevent rejection would also be sufficient to prevent T1DR. Later reports suggested T1DR could occur in unrelated SPK transplant recipients of deceased donor organs, despite HLA mismatch and immunosuppression. Our evaluation of SPK recipients who developed T1DR, like our SPK transplant program, has also evolved overtime. The initial studies of selected cases with possible T1DR revealed that the pancreas transplant was usually of normal size by ultrasound or computed tomography (CT) scan imaging of the abdomen. This finding helped to differentiate T1DR from chronic pancreas transplant rejection, in which case the pancreas transplant tended to appear small, shrunken, and fibrotic on imaging studies. The next level of diagnostic evaluation involved biopsy of both the pancreas transplant and kidney transplant which provided material for histological study; this revealed that the underlying pancreas transplant lesion was in fact insulitis, and not rejection, and that the kidney transplant biopsy also did not show rejection. The demonstration of insulitis in our early experience lead to collaboration resulting in the testing of autoantibodies associated with T1D risk, such as those against GAD65, IA2, and ZnT8. Further collaboration added the testing of autoreactive antigen-specific T cells which could be assessed via tetramer studies. The pancreas transplant biopsies were performed via open surgical exploration, with collection of peripancreatic transplant tissues, including lymph nodes. The pancreas transplant, peripancreas transplant tissues, and peripheral blood were all assayed for autoreactive T cells.14,15 As we have studied this field over the past couple of decades, we have made observations in the following areas: (1) remodeling/transdifferentiation of the pancreas transplant, (2) treatment of autoimmune recurrence, (3) cell-mediated responses in T1DR (autoreactive T cell-­ mediated beta cell destruction experimentally in  vivo, together with a possible case of incipient T1DR), and (4) re-transplantation of the pancreas transplant following autoimmune recurrence. Remodeling/transdifferentiation in pancreas transplant of T1DR One of our earliest observations was that, similar to recent onset T1D, simultaneous pancreas-kidney transplantation (SPKT) recipients with T1DR consistently showed that insulin-staining beta cells were identifiable in the pancreas transplant.16 At the time this was surprising, since it was believed that once an SPK transplant recipient developed severe hyperglycemia, all of its islet cell mass was likely to be destroyed. In another one of our early studies, we obtained pancreas transplant biopsies from nine patients who exhibited signs of T1DR.17 These patients were diagnosed based on the following criteria: (1) hyperglycemia requiring insulin therapy; (2) clinical

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symptoms of diabetes in the presence of unchanged pancreas transplant exocrine function and kidney transplant function; (3) autoantibodies and/or autoreactive T cells in the circulation; (4) insulitis and/or beta cell loss at biopsy; and (5) reduction in c-peptide serum levels. Interestingly, we identified the existence of insulin-­ positive ductal cells confirmed by CK-19 colocalization in the ducts of the transplanted pancreas specifically in the SPK patients with recurrent autoimmunity and diabetes17 (Fig.  2). The patient with the most severe beta cell destruction and complete loss of c-peptide secretion at the time of biopsy was the one with the highest number of ducts containing insulin-positive cells. A consistent feature observed in our SPK transplant recipients with T1DR was that in those ducts that contained insulin-­ positive cells, most, although not all, ductal cells were insulin-positive. None of these ductal cells stained for glucagon. In contrast, in one patient who was normoglycemic, without beta cell loss, who did have clear evidence of ongoing autoimmunity there was virtually no evidence for insulin-positive staining in the ductal cells. So, the presence of both hyperglycemia and autoimmunity may be critical for triggering insulin synthesis in ductal cells. Despite the presence of insulin identified within the ductal cells, there was no identifiable c-peptide in circulation, so these cells are able to synthesize, but not secrete insulin. Furthermore, we found that insulin-positive, CK-19 positive cells can also stain for Ki-67 (Fig. 3), suggesting that these cells are capable of replication.17 This was observed in the patient with the most severe beta cell destruction suggesting a possible link between the replication of insulin-positive ductal cells and the severity of beta cell loss/hyperglycemia. Ki-67 staining was not seen in the surviving beta cells in the islets of any SPK patients with T1DR.17 Our findings suggest that ductal cells participate in beta cell regenerative processes occurring in the transplanted human pancreas in the context of hyperglycemia and T1DR. Perhaps, these may be critical stimuli to trigger pancreas remodeling mechanisms. Dissecting the mechanisms involved in the remodeling processes could lead to therapeutic implications for both (1) T1DR and (2) possibly regenerative medicine research involving the application of stem cell differentiation into insulin-secreting islet cells. Treatment of T1DR: Evidence for return of memory T cells in T1DR It is possible that the pancreas transplant portion of the SPKT may mimic an antigen booster immunization, with T1DR as an anticipated outcome. Because T1D is characterized by a lack of islet cells, the pancreas transplant with its full complement of islet cells may induce a recall memory response, reactivating memory cells that have been quiescent since the onset of T1D many years previously. This response, from the development

or increase in levels of autoantibodies, to the presentation with hyperglycemia due to T1DR, may take years to become manifest. Although our sample size is limited, our studies of TCR clonotypes revealed that GAD65 autoreactive CD4 T cells expressing the same V-beta chains (5.1 and 9) and identical or similar CDR3 sequences, reappeared after the development of T1DR, despite the fact that all SPK patients are receiving immunosuppression.18,19 The persistence of autoreactive T cells vs the same autoantigen, expressing the same or similar TCR V-beta chains, is consistent with a memory response associated with T1DR in our SPKT recipients. We have detailed time courses of a number of our SPKT recipients who have developed T1DR. Specifically, we initially identified two patients (and have added a third) who developed T1DR, several years following SPKT, in whom there was evidence for persistent c­-peptide circulating in the serum in response to a mixed meal challenge, and the pancreas transplant biopsy showed the presence of islet cell insulin staining. They both experienced a rise in levels of autoantibodies and demonstrated insulitis on pancreas transplant biopsy.18 The T cell infiltrate was found to include both CD4 and CD8 positive T cells. The cardinal findings of T1D including (1) insulitis on pancreas transplant biopsy, (2) the identification of autoantibodies in the peripheral blood, and (3) the presence of autoreactive T cells in the peripheral blood were identified in both patients. The first patient developed hyperglycemia 5-years after SPK transplantation. Retrospectively both GAD65 and IA-2 levels persisted following transplantation, and both levels rose prior to hyperglycemia (Fig. 4). There was also evidence for autoreactive GAD65 CD4+ T cells in the peripheral blood just prior to treatment, about 1-year after return to hyperglycemia. This SPKT recipient was treated with our induction therapy regimen for SPK transplants that is directed against mostly T cells, including thymoglobulin (1 mg/kg, times five doses) and anti-CD25 monoclonal antibody (daclizumab, 1 mg/kg, times two doses). Subsequently, autoantibody levels fluctuated, autoreactive T cells were undetectable for nearly 1 year, and fasting c-peptide levels rose over the ensuing 6 months. However, the patient remained on insulin, and fasting c-peptide levels fell after the reappearance of GAD65 autoreactive T cells in the peripheral blood and were undetectable 3-years after treatment.18 Of note, in these early studies, when CD4-positive autoreactive T cells were identified, a significant population was noted to express membrane markers of memory (CD45RO). The second patient became hyperglycemic 9 and one-half years after SPK transplantation. Anti-GAD65 and anti-IA2 autoantibodies became detectable about 6 years after transplantation, and hyperglycemia ensued three and one-half years later18 (Fig. 5). CD8+ T cells that reacted to IGRP (islet-specific glucose-6 p ­ hosphatase

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FIG. 2  Insulin+ cells in pancreatic ducts. Insulin protein was demonstrated by immunohistochemistry in pancreas transplant biopsies from 6 SPK patients with recurrent autoimmunity (A–N). Patients 1–5 (A–M), who had developed recurrence of diabetes, had the greatest number of ductal cells stained for insulin. (A) Patient 1, first biopsy; infiltrating lymphocytes are seen surrounding a duct containing insulin+ cells. (B and C) Patient 1, second biopsy, serial sections stained for insulin and isotype control, respectively. Staining is weak (B), but distinct and clearly above background (C). (D) Patient 1, second biopsy, larger duct stained for insulin; staining is present also on the nonluminal side in many cells. (E and F) Patient 1, second biopsy, ducts with intense insulin staining, and (G) patient 1, a few ducts and some smaller duct-like structures (lower right corner) stained for insulin. (H) Biopsy of patient 2, showing larger ducts and some smaller duct-like structures stained for insulin. (I) Biopsy of patient 3, insulin staining in a duct (arrow) is weaker than in the nearby islet. Some weaker staining is seen in the acinar tissue, possibly involving smaller duct-like structures. (J) Pancreas transplant biopsy of patient 4, showing three islets, one of which was heavily infiltrated by lymphocytes. (K and L) Higher magnification (~2×) of details (J) to better visualize ducts containing insulin+ cells (arrows). Some weaker and less distinct staining is seen in the acinar tissue, possibly involving smaller duct-like structures. (M) Pancreas transplant biopsy of patient 5; not all ductal cells stained for insulin (arrows); staining is also present on the nonluminal side in many cells. (N) Insulin+ cells (arrow) were rarely seen in a minority of ducts in patient 6; he had not yet developed significant insulitis and beta cell destruction, and was normoglycemic. (O) Insulin+ cells (arrow) were also rarely seen in control pancreases. Some single insulin+ cells were also seen in the acinar tissue. Objective lens: 63× (A, D, F, G), 100× (B, C, E), 40× (H, I, M, N, O), 20× (J, K, L are details from J shown at magnification, ~2×). Reproduced from Martin-Pagola A, Sisino G, Allende G, Dominguez-Bendala J, Gianani R, Reijonen H, Nepom GT, Ricordi C, Ruiz P, Sageshima J, Ciancio G, Burke GW, Pugliese A. Insulin protein and proliferation in ductal cells in the transplanted pancreas of patients with type 1 diabetes and recurrence of autoimmunity. Diabetologia 2008;51:1803–1813. https://doi. org/10.1007/s00125-008-1105-x.

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One year later, autoreactive, IGRP-specific CD8+ memory T cells were again identified in the peripheral blood, and c-peptide levels became undetectable.18 A third SPK transplant recipient was diagnosed with T1DR, and treated with an immunosuppressive regimen that incorporated all the previous therapies, anti-T cell (thymoglobulin, and anti-CD25 monoclonal antibody), and anti-B cell therapy (rituximab), as well as adding plasmapheresis, and intravenous immunoglobulin (IVIg). Plasmapheresis and IVIg were added in an attempt to modulate levels of autoantibodies. In a similar fashion to our first two SPK recipients with T1DR, this patient also experienced an initial rise in fasting ­c-peptide levels, but once the autoreactive memory T cells returned to the peripheral blood, the c-peptide levels were inexorably lost (data not shown).

FIG. 3  Colocalization analysis of insulin, CK-19 and Ki-67. (A) The absence of colocalization of insulin and CK-19 in control pancreas 15. A Ki-67+ cell is shown outside the duct. (B) Colocalization of insulin, CK-19, and Ki-67 in the pancreas transplant biopsy of patient 1. Colocalization of insulin with CK-19 was seen, and one cell in the duct also stained for Ki-67. Objective lens: 20× (A) and 40× (B). Reproduced from Martin-Pagola A, Sisino G, Allende G, Dominguez-Bendala J, Gianani R, Reijonen H, Nepom GT, Ricordi C, Ruiz P, Sageshima J, Ciancio G, Burke GW, Pugliese A. Insulin protein and proliferation in ductal cells in the transplanted pancreas of patients with type 1 diabetes and recurrence of autoimmunity. Diabetologia 2008;51:1803–1813. https://doi.org/10.1007/s00125-008-1105-x.

c­atalytic subunit-related protein, an islet cell autoantigen) were identified in the peripheral blood prior to treatment with immunosuppression. In view of the ultimate lack of efficacy using our T-cell-directed regimen of thymoglobulin and anti-CD25 for our first patient, we decided to add anti-B cell therapy to this, and so a single dose of rituximab (375 mg/m2, anti-CD20, a marker on B cells, monoclonal antibody) was also used for treatment. We reasoned that in addition to our anticipated T cell effect, the addition of rituximab could potentially affect B cells, and possibly autoantibodies. After this treatment, the autoantibodies again fluctuated, and the autoreactive CD8+ T cells fell during the period of 1 year. The fasting c-peptide levels rose initially, similar to our first patient, although this patient also remained on insulin therapy.

Cell-mediated responses in T1DR Autoreactive T cell-mediated beta cell destruction experimentally in vivo In  vivo studies demonstrated that these CD4+ autoreactive T cells from our SPK patients with T1DR could mediate beta cell destruction of human islets.18 Specifically, autoreactive CD4+ T cells isolated from our SPK transplant recipients with T1DR were co-­ transplanted with human islet cells from a different donor (HLA-mismatched) under the kidney capsule of an immunodeficient mouse. Controls included mice receiving human islets alone, or human islets with irrelevant human T cells from the same patients. After 10–16 days the kidneys were removed and the transplanted islets were stained for insulin and glucagon, as well as staining with hematoxylin and eosin. The control grafts demonstrated good islet architecture and an abundance of insulin and glucagon staining. In contrast, those islets from transplants that had received the autoreactive CD4+ T cells showed severely disrupted islet architecture and much reduced insulin staining (Fig.  6). In one of the experiments in which the mice were rendered diabetic with streptozotocin prior to islet transplantation, diabetes was reversed in the control mouse that received the islets alone, and the mouse that received islets with irrelevant T cells. However, in the recipient of islets with the autoreactive CD4+ T cells, normoglycemia was never achieved (Fig.  7). This experiment provided a physiologic correlate to the in vivo identified destruction of the human islets by the GAD65-autoreactive CD4+ T cells.18 A possible case of incipient T1DR In addition to identifying CD4 and CD8 positive, autoreactive T cells in the peripheral blood we identified autoreactive T cells in the pancreas transplant and the peripancreas transplant lymph nodes in two separate, distinct clinical situations. The first involved

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FIG. 4  Clinical course, autoimmunity assessment and biopsy in patient 1 (Ref. 18). Patient 1 was a 41-year-old Caucasian male [HLA A2/A3, B57/B60, DR4 (DRB1*0405)/DR6] who developed type 1 diabetes at age 7. He received an SPK transplant from an HLA A2/A30, B41/B60, DR4/ DRX donor at age 32. The transplant reversed diabetes, but the patient returned to insulin dependence 5 years later while kidney and exocrine pancreas allografts had normal function. Panel A: Autoantibody levels prior to transplant and on follow-up. The patient had GAD and IA-2 autoantibodies prior to transplantation, which persisted despite immunosuppression and titers increased on follow-up. Colour-matched, horizontal lines represent the cut-off level for each autoantibody. For all autoantibodies, a value>1 denotes a positive result. Panel B: Pancreas transplant biopsy stained as labeled, obtained about 6 months after the recurrence of hyperglycemia. Sections were stained as labeled. Insulitis and ß-cell loss are shown. Panel C: Serum C-peptide levels and % of GAD tetramer-positive T-cells in the CD4 T-cell population from the time of hyperglycemia recurrence. C-peptide was still detectable at diagnosis, confirming the function of residual ß-cells observed at biopsy. Autoreactive T-cells were detected at the time of biopsy, about 6 months after the recurrence of hyperglycemia on two samples, and again at several time points about 1 year following treatment. The horizontal blue line represents the cutoff of the tetramer assay (0.25%). Panel D: Flow cytometry plots demonstrating GAD-autoreactive CD4 T-cells. The numbers above the plots identify the same sample on panel C. Tetramer staining with irrelevant peptide was <0.1% (not shown). Reproduced from Vendrame F, Pileggi A, Laughlin E, Allende G, Martin-Pagola A, Molano RD, Diamantopouls S, Standifer N, Geubtner K, Falk BA, Ichii H, Takahashi H, Snowhite IV, Chen Z, Mendez A, Chen L, Sageshima J, Ruiz P, Ciancio G, Ricordi C, Reijonen HK, Nepom GT, Burke GW III, Pugliese A. Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T cells. Diabetes 2010;59:947–957. Copyright © American Diabetes Association (ADA).

i­dentification of proinsulin-staining autoreactive T cells in the lymph nodes of a pancreas transplant in a patient who was euglycemic two and one-half years after SPK transplantation, but found to have minimal insulitis on pancreas transplant biopsy as well as normal insulin staining of the beta cells in the pancreas transplant.20 This

patient was also noted to be seroconverting for GAD65 and IA2 autoantibodies at the time of this biopsy and was found to have autoreactive T cells in the pancreas transplant, peripancreas transplant lymph nodes, and in the peripheral blood. This finding raised the ­possibility, consistent with traditional thinking, that the initial

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FIG. 5  Clinical course, autoimmunity assessment, and biopsy in patient 2.18 Patient 2 is a Caucasian female (HLA A2/A24, B44/B56, DR5/DR9) who developed type 1 diabetes at age 8. She received an SPK transplant from an HLA A2/A3, B7/B14, DR7/DR9 donor at age 30. Her pancreas transplant successfully reversed diabetes. After approximately 9 years, the patient developed hyperglycemia requiring insulin therapy while the function of the kidney and exocrine pancreas allografts remained unchanged. Panel A: Autoantibody levels prior to transplant and on follow-up. The patient converted to GAD and IA-2 autoantibody positivity 6 years after transplantation. Hyperglycemia ensued 3.5 years after autoantibody conversion. Panel B: Pancreas transplant biopsy stained as labeled, obtained about 7 months after the recurrence of hyperglycemia. There was evidence for insulitis and ß-cell loss. Panel C: C-peptide levels from the time of hyperglycemia recurrence and percentage of IGRP tetramer-­positive T-cells in the CD8 T-cell population. The horizontal blue line represents the cutoff of the T-cell assay (0.1%). Percentage of cells plotted is the specific staining value shown in panel D minus the background staining with control peptide. Circulating CD8 T-cells reacting against IGRP were found in sample obtained at the time of biopsy and again about 1 year following treatment. Panel D: Flow cytometry plots showing IGRP-specific, autoreactive CD8 T-cells. Staining with tetramers loaded with a control peptide yielded 0.1% background staining levels, gating on PBMC (not shown). The numbers above the plots identify the IGRP T-cell measurements in panel C, thus corresponding to the samples measured closest to the onset of hyperglycemia and over 1 year after treatment. Reproduced from Vendrame F, Pileggi A, Laughlin E, Allende G, Martin-Pagola A, Molano RD, Diamantopouls S, Standifer N, Geubtner K, Falk BA, Ichii H, Takahashi H, Snowhite IV, Chen Z, Mendez A, Chen L, Sageshima J, Ruiz P, Ciancio G, Ricordi C, Reijonen HK, Nepom GT, Burke GW III, Pugliese A. Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T cells. Diabetes 2010;59:947–957. Copyright © American Diabetes Association (ADA).

a­ utoimmune event may have been the stimulation of autoreactive T cells in the peripancreas transplant lymph node(s). This may have led to egress of the stimulated autoreactive T cells from the lymph nodes, with their eventual appearance in the peripheral blood, prior to returning to the pancreas transplant, where initiation of the beta cell infiltrative process began. The localization

of these autoreactive CD4+ T cells to the lymph nodes, peripheral blood and pancreas, provides possible insight into the pathogenesis of T1DR, and possibly T1D. This finding influenced our approach to other cases, specifically in the instance of a patient with recurrence of autoimmunity who later received a second pancreas transplant,16 described in the following section.

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FIG. 6  In vivo assessment of the autoreactive potential of GAD-autoreactive CD4 T-cells from patient 1.18 A peripheral blood sample (sample no. 10, Fig. 4C and D) from patient 1 yielded a very strong CD4 T-cell response to GAD; 7% of the CD4 T-cells were GAD autoreactive after in vitro stimulation and stained specifically with the DRB1*0405-GAD 555–567 tetramer (A). Approximately 15,000 tetramer-positive, GAD-autoreactive CD4 T-cells were purified by fluorescence-activated cell sorting and co-transplanted with human islets (1300 islet equivalents), freshly isolated from an unrelated, deceased donor [HLA-A30, A33, B42, B70, DR8, DR17(3)], under the kidney capsule of a nondiabetic immunodeficient mouse. Control mice received islets with 15,000 CD4 T-cells from the same patient, which were stimulated with the HA control peptide and sorted after staining with a DRB1*0405-HA tetramer (B) or islets alone (C). H&E and insulin and glucagon stains representing the same areas of the graft reveal damaged islets and loss of insulin staining in the graft that received GAD-specific CD4 T-cells (A). Normal graft morphology and hormone staining patterns are seen in control mice receiving HA-specific CD4 T-cells (B) or islets alone (C). Reproduced from Vendrame F, Pileggi A, Laughlin E, Allende G, Martin-Pagola A, Molano RD, Diamantopouls S, Standifer N, Geubtner K, Falk BA, Ichii H, Takahashi H, Snowhite IV, Chen Z, Mendez A, Chen L, Sageshima J, Ruiz P, Ciancio G, Ricordi C, Reijonen HK, Nepom GT, Burke GW III, Pugliese A. Recurrence of type 1 diabetes after simultaneous pancreas-­ kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T cells. Diabetes 2010;59:947–957. Copyright © American Diabetes Association (ADA).

Re-transplantation of the pancreas for T1DR We describe an SPK transplant recipient who developed severe T1DR (Ref. 17, which shows ductal cells staining positive for insulin and, patient #3, Ref. 18), and underwent re-transplantation of the pancreas. This patient was euglycemic for 5 years after transplantation, and then became hyperglycemic (insulin-dependent), with a functioning kidney transplant and persistent pancreas transplant exocrine function (urine amylase). Serum c-peptide was lost, and the pancreas transplant biopsy showed glucagon, but no insulin staining18 (Fig.  8). There was also some, albeit minimal, insulitis, suggesting that the active phase of recurrent disease had passed. Thus, this patient was not considered to be a candidate for possible therapy to recover islet cell

function. Instead, a second pancreas transplant was performed 1 year after the presentation with hyperglycemia. Levels of autoantibodies, both GAD65 and ZnT8 rose, approximately 3 months prior to hyperglycemia, and were gradually falling at the time of re-transplantation. Induction immunosuppression included thymoglobulin, anti-CD20 (daclizumab), and rituximab. This patient was also treated with plasmapheresis and IVIg. GAD65 autoantibody levels were unaffected by plasmapheresis, but the levels of ZnT8 appeared to have been reduced. The tail of the original pancreas transplant was removed at the time of the second pancreas transplant surgery, in order to gain surgical access for the vascular anastomoses. Since the head of the pancreas was not in the way from a surgical standpoint, it was left in place. This

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Nonfasting glycemia (mg/dL)

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T cells, in the pancreas transplant and peripancreatic transplant lymph nodes and tissues. We hypothesize that removing the entire first pancreas transplant and peripancreas transplant tissue may remove a key population of autoreactive T cells, and perhaps help prevent future recurrence in the context of re-transplantation of the pancreas for T1DR.

Control GAD CD4 T cells Polyclonal CD4 T cells

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FIG.  7  In  vivo assessment of the autoreactive potential of GAD-

autoreactive CD4 T-cells from patient 3.18 Immunodeficient mice with streptozotocin-induced diabetes were transplanted with human islets (2000 islets equivalents) from an HLA-A1, A29, B8, B44, DR17(3)/DRX donor, which had been cultured for 24 h, and T-cells purified from blood obtained from patient 3 at 9.5 years of follow-up. Specifically, we purified and co-transplanted islets with 6000 tetramer-positive, GADautoreactive CD4 T-cells (6% of the CD4 T-cells). Control diabetic mice received islets and 30,000 CD4 T-cells after stimulation with the OspA control peptide or islets alone. There was no response to this negative control peptide. Thus, this was a polyclonal T-cell population, which could have included alloreactive T-cells. CD25 staining confirmed that these T-cells had been activated in vitro. On metabolic follow-up, both control mice reversed their diabetes after islet transplantation, while the mouse that received the GAD-autoreactive CD4 T-cells remained hyperglycemic for the entire duration of the experiment. Control mice reverted to diabetes when the grafts were removed by nephrectomy (arrow) after 2 weeks. Reproduced from Vendrame F, Pileggi A, Laughlin E, Allende G, Martin-Pagola A, Molano RD, Diamantopouls S, Standifer N, Geubtner K, Falk BA, Ichii H, Takahashi H, Snowhite IV, Chen Z, Mendez A, Chen L, Sageshima J, Ruiz P, Ciancio G, Ricordi C, Reijonen HK, Nepom GT, Burke GW III, Pugliese A. Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T cells. Diabetes 2010;59:947–957. Copyright © American Diabetes Association (ADA).

­ atient was noted to develop T1DR again 3 years later in p the second pancreas transplant, and expressed a similar autoimmune pattern to that which appeared just prior to the original development of T1DR. Levels of GAD65 and ZnT8 autoantibodies rose sharply just ahead of the development of hyperglycemia, and GAD65-specific CD4+ T cells became detectable in the peripheral blood. Importantly, autoreactive T cells were identified in the residual portion of the head of the original pancreas transplant as well as in lymph node tissue surrounding the head of the original pancreas transplant18 on reexploration. This patient ultimately experienced severe rejection of the second pancreas transplant, after exhibiting evidence of reactivation of islet autoimmunity consistent with T1DR. This case of re-transplantation of the pancreas for T1DR, and the previous description of incipient T1DR shed light on the importance of the identification of autoreactive CD4+ T cells, most of which were memory

We evaluated islet autoimmunity overtime, prior to and after transplantation, retrospectively in 223 of our SPKT recipients. We found that (1) conversion to autoantibody positivity and (2) higher numbers of autoantibodies, up to three autoantibodies, were significant risk factors for the development of T1DR21 (Fig. 9). Matching of donor and recipient for DR3 and/or DR4 also increased the risk of T1DR (OR = 3.5, P = .02). Approximately 5% of our SPKT recipients develop T1DR, and generally present with hyperglycemia about 5 years after transplantation. T1DR is not typically observed in the immediate posttransplant period and cannot be simply considered to be triggered by the reintroduction of beta cells and autoantigens with the transplant.21 In fact, 43% of SPK recipients were autoantibody positive at the time of transplantation (Fig.  10). This did not appear to influence the outcome, however, since most autoantibody levels either converted to negative (about 20%), or remained largely stable and in the low range overtime (persistent, about 60%). We observed increased risk of T1DR only in recipients who experienced autoantibody conversion (from negative pretransplant to positive after transplant) or the acquisition of additional autoantibodies after transplantation (about 20%). The range of patients presenting with T1DR has been from 5 to 20 years following SPK transplantation. Thus, in our cohort we may identify 1 or 2 patients per year developing T1DR for evaluation and possible therapy. A similar proportion of SPKT recipients develop chronic rejection of the pancreas, an allogeneic immune response, losing insulin secretion and also returning to insulin therapy, underscoring the clinical importance of T1DR, which affects a similar proportion of SPK transplant recipients. These SPK recipients with chronic rejection will also lose urinary amylase, consistent with the loss of both endocrine and exocrine pancreas transplant function, in contradistinction to those patients with T1DR, in whom pancreas transplant exocrine function, assessed by urine amylase measurements, is typically preserved. While T1DR occurs in the setting of an allogeneic donor pancreas transplant, and in the context of immunosuppressive medication, the pattern of autoantibody development, along with the insulitis seen on biopsies, and time course of glycemic derangement subsequent to

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FIG. 8  Clinical course, autoimmunity assessment, and biopsy in patient 3.18 Patient 3 is a 38-year-old Caucasian male [HLA A26/A30, B38/ B58, DR3/DR4 (DRB1*0402)] who developed type 1 diabetes at age 12. He received an SPK transplant from an HLA-A23/A33, B7/B52, DR2/ DR10 donor at age 27. The pancreas transplant successfully reversed diabetes. Five years later the patient developed hyperglycemia requiring insulin therapy with unchanged function of the kidney and exocrine pancreas allografts. Panel A: Autoantibody levels prior to transplant and on follow-up. Colour-matched blue and black horizontal lines represent cutoffs for GAD/IA-2 and ZnT8 autoantibodies, respectively. The patient had been autoantibody-negative prior to transplant and for almost 5 years on follow-up, but converted to GAD and ZnT8 autoantibody positivity about 3 months before the recurrence of hyperglycemia. At the time, there was a sharp rise in ZnT8 autoantibodies, shortly thereafter followed by a similar rise in GAD autoantibody levels, peaking at levels which were 40-fold and 10-fold higher than the upper limit of normal, respectively. Inset: Hormone stains in the first pancreas transplant biopsy obtained at re-transplantation demonstrate ß-cell loss. Panel B: Serum C-peptide levels and percentage of GAD tetramer-positive T-cells in the CD4 T-cell population from the time of hyperglycemia recurrence. Patient 3 had no residual C-peptide secretion in the fasting state and no response to a sustacal meal test (not shown) at the onset of hyperglycemia. C-peptide secretion was restored by re-transplantation but was lost again after rejection of the second pancreas transplant. GAD-specific autoreactive CD4 T-cells were first studied in the sample obtained before the immunosuppression required for the second transplant. Autoreactive T-cells became undetectable following immunosuppression but eventually rebounded and were detected on multiple occasions. The horizontal blue line represents the cutoff of the tetramer assay (0.25%). Panel C: Flow cytometry plots demonstrating strong responses of GAD autoreactive, CD4 T-cells. Numbers above the plots correspond to those in panel B. Tetramer staining with irrelevant peptide was <0.1% (not shown). Panel D: Biopsy of the second pancreas graft showing rejection. CD4 infiltrates are seen near residual insulin stained areas. Reproduced from Vendrame F, Pileggi A, Laughlin E, Allende G, Martin-Pagola A, Molano RD, Diamantopouls S, Standifer N, Geubtner K, Falk BA, Ichii H, Takahashi H, Snowhite IV, Chen Z, Mendez A, Chen L, Sageshima J, Ruiz P, Ciancio G, Ricordi C, Reijonen HK, Nepom GT, Burke GW III, Pugliese A. Recurrence of type 1 diabetes after simultaneous pancreas-­ kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T cells. Diabetes 2010;59:947–957. Copyright © American Diabetes Association (ADA). A.  Whole pancreas allo-transplantation

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pancreas transplantation, suggest that T1DR occurs in a manner that closely resembles the mechanism of autoimmunity in T1D in the native pancreas. Interestingly, studies of native T1D in pediatric populations reflect similar time courses for autoantibodies as that seen in our patients.22,23 Another cause of return to insulin therapy after SPKT is related to neither auto- nor allogeneic immunity, but is likely due to the development insulin resistance related to weight gain and side effects of the immunosuppressive medications.

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transplant, based on which we determined overall antibody patterns (negative, conversion, persistence) in 223 SPK recipients. Reproduced from Vendrame F, Hopfner YY, Diamantopoulos S, Virdi SK, Allende G, Snowhite IV, Reijonen HK, Chen L, Ruiz P, Ciancio G, Hutton JC, Messinger S, Burke GW III, Pugliese A. Risk factors for type 1 diabetes recurrence in immunosuppressed recipients of simultaneous pancreas kidney transplants. Am J Transplant 2016;16(1):235–245. doi: 10.1111/ajt. 13426. Copyright © 1999–2018 John Wiley & Sons, Inc. All rights reserved.

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(C) FIG. 9  Kaplan-Meier analysis of HG-T1DR free survival according to the autoantibodies on follow-up. (A) Autoantibody positivity: positive vs negative, HR = 14.53, P = .0005. (B) Number of autoantibodies: overall, P = .0001; 1 vs 0, HR = 3.53, P = .2672; 2 vs 0, HR = 14.60, P = .0005*; 3 vs 0, HR = 53.88, P < .0001*; 2 vs 1, HR = 5.65, P = .0131**; 3 vs 1, HR = 17.04, P < .0001*; 3 vs 2, HR = 3.00, P = .0261**); P value *was or **was not significant after correction for multiple comparisons. (C) Autoantibody conversion: overall, P < .0001; persistence vs negative, HR = 1.88, P = .6486; conversion vs negative, HR = 27.11, P < .0001*; conversion vs persistence, HR = 15.57, P = .0003*; *significant P values after correction for multiple comparisons. Reproduced from Vendrame F, Hopfner YY, Diamantopoulos S, Virdi SK, Allende G, Snowhite IV, Reijonen HK, Chen L, Ruiz P, Ciancio G, Hutton JC, Messinger S, Burke GW III, Pugliese A. Risk factors for type 1 diabetes recurrence in immunosuppressed recipients of simultaneous pancreas kidney transplants. Am J Transplant 2016;16(1):235–245. doi: 10.1111/ajt. 13426. Copyright © 1999–2018 John Wiley & Sons, Inc. All rights reserved.

Therapy for T1D Over the course of four decades, there have been numerous clinical trials to treat patients with newly diagnosed T1D using various agents including (1) anti-CD3 monoclonal antibody,24,25 (2) GAD65 antigen,26 (3) rituximab (anti-CD20 monoclonal antibody),27 (4) abatacept (soluble fusion protein binding CD80 and CD86),28 and (5) alefacept (a CD2-directed LFA-3/Fc fusion protein).29 The preclinical research that led to the selection of these agents was mostly based on the experimental data from the NOD mouse, which is considered a close surrogate for T1D in humans.30–33 The results of these studies have been largely disappointing. While some preservation of insulin secretion has been seen for a limited period of time, full disease remission and improvement of insulin secretion have not been typically observed. However, there was prolonged preservation of islet cell function

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Introduction

[c-peptide secretion/area under the curve during an mixed-meal tolerance test (MMTT)] at 2 years posttherapy in patients treated with alefacept,29 and in a subset of patients treated with anti-CD3.34

T1DR therapy at the Miami Transplant Institute In our experience with T1DR, we have used combinations of thymoglobulin, anti-CD25 monoclonal antibody, steroids, rituximab, IVIg, and plasmapheresis to treat patients with biopsy proven T1DR.16 In our first two reported cases, the patients experienced a transient increase in fasting C-peptide; however, upon return of memory autoreactive CD4-positive or CD8-positive T cells to the peripheral blood, c-peptide secretion was ultimately lost.18,19 Since our early studies, we have used other medications including alefacept and ustekinumab, both of which have been used to treat patients with psoriasis,35 for therapy of our patients with T1DR. As noted above, alefacept has been used therapeutically in a trial of new onset T1D,19 and a trial using ustekinumab for new onset T1D is ongoing in British Columbia. Thus, the concept of translating therapeutic agents useful in dermatologic autoimmunity to a similar role in T1D, and hence T1DR, has precedence. Prior to treatment for T1DR, our SPK transplant recipients undergo metabolic testing with an MMTT in order to establish baseline c-peptide, insulin, and proinsulin responses. In our clinical experience, the c-peptide response curve in patients with T1DR is usually flat, showing c-peptide levels between 1 and 2 ng/mL over the time course (2–4 h), without significant increment despite the fact that glucose levels are in the diabetic range. Our experience with alefacept included therapy for a patient in whom CD2-positive memory autoreactive T cells were identified. This was an example of the identification of critical memory markers—both CD2 and CD45RO—on islet lymphocytic infiltrates and on circulating peripheral blood autoreactive T cells that could be monitored. This patient received a 3-month course of alefacept. During treatment autoreactive CD8-positive T cells fell to nearly undetectable levels,36 while hemoglobin A1c fell from 10 to 6 and, the Lantus requirements were cut in half, from 20 to 10 units/day. However, alefacept was taken off the market and an anticipated second 3-month course of alefacept could not be given. Similar to our experience, the demonstrated sustained clinical and immunologic effects of alefacept reported in patients with recently diagnosed T1D29 will have to be further explored when a biosimilar becomes available. Currently, there are no available agents to effectively treat lymphocytic infiltrates characterized by memory T cells in T1D or T1DR. Another intervention included the use of ustekinumab, which has anti-IL 12 and anti-IL 23 (IL-17) features35 based on binding to the p40 portion of the r­espective

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receptor. A 3-month course of ustekinumab was given to a patient who experienced T1DR nearly 20  years post-SPKT. This patient had evidence of Th1 infiltrate on pancreas transplant biopsy documented by staining for T-bet, together with significant numbers of insulin-­ positive islets; there was some residual but otherwise flat and impaired c-peptide secretion in response to MMTT. This patient was treated with the lower dose (45 mg) of ustekinumab to limit the likelihood of overimmunosuppression, since he remained on maintenance immunosuppression to protect the kidney transplant. Metabolic testing after the 3-month course of ustekinumab showed no change in c-peptide/glucose ratios, hemoglobin A1c or insulin requirements, and this patient has remained on insulin since that time. A study in new onset diabetes was recently undertaken in British Columbia37 using ustekinumab at two doses, 45 and 90 mg. The interim analysis (available through NCT02117765) demonstrated a mean reduction in c-­peptide area under the curve of 0.1 pmol/mL and mean 50% reduction in peripheral blood Th 17 cells only in the group that received 90 mg dosing. This was significantly better than what was seen in the cohort that received 45 mg. This result confirmed the importance of using the higher, that is, 90 mg, dosing regimen of ustekinumab in the context of diagnosis of T1D. Interestingly, risankizumab, a monoclonal antibody which binds to the p19 component of the IL-23 receptor and does not impact the IL-12 receptor (p35, p40), has been demonstrated to be significantly more effective from a therapeutic standpoint for moderate to severe plaque psoriasis than ustekinumab.38 Perhaps risankizumab could have potential therapeutic utility in our unique clinical context of T1DR. We are currently involved in expanding our search for involvement of Th17 lymphocytes in the pancreas transplant biopsies of our patients with T1DR to determine whether the use of an IL-23/17-specific agent could be helpful.

Theoretical considerations CXCR3 as a potential target in T1DR In our recent work, CXCR3, a chemokine receptor, was identified on islet-infiltrating T cells in the pancreas transplant of a patient who experienced T1DR. This was associated with a higher level of autoreactive, memory T cells identified in the peripheral blood that also stained for CXCR3.39 CXCR3 has been identified in the past as being responsible for mediating T cell infiltration in insulitis in the NOD mouse.40 We also identified autoreactive memory CD4 positive T cells that stained for CXCR3 in the peripancreatic transplant lymph nodes. This raises the possibility that therapy directed at CXCR3, the target of cytokines CXCL9 and CXCL10, could potentially be therapeutic for T1DR and possibly T1D. Moreover, since CXCR3 autoreactive memory T cells can be found in the

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peripheral blood of these patients with T1DR, this can likely be used as a peripheral blood biomarker in the context of CXCR3-specific therapy. This is all the more relevant in view of the development of a monoclonal antibody to CXCR3 currently for clinical use in psoriasis. CXCR3 in alopecia areata and AA as a model of T1DR Borrowing again from dermatologic models of autoimmunity, studies in patients with alopecia areata (AA) demonstrated CXCR3 on infiltrating T cells in skin biopsies of these lesions. This led to translational studies in a mouse model using an antibody to CXCR3, which resulted in improvement in this mouse model.41 Transcriptional profiling led to the identification of a panel of inflammatory/cytokine markers including the interferon pathway42 and genes involved with graft vs host disease, T1D, allograft rejection, cell adhesion, antigen processing and presentation, and chemokine signaling, in the skin biopsies of patients with AA. Interestingly, similar gene profiles were identified in the pancreas of patients with T1D and in the pancreas transplant of patients with T1DR.43 These findings in alopecia lead to the use of JAK/Stat inhibitors44–46 including tofacitinib47 and subsequently ruxolitinib.48 A small study with ruxolitinib showed higher proportions of patients who responded to therapy along with an apparent ­longer-lasting effect than with tofacitinib.48 These studies are currently being expanded and generating longer follow-up. This raises the possibility that similar mechanisms of action may be at play in both AA and T1DR, since both involve the presence of CXCR3 on infiltrating cells, as well as the generation of similar gene expression profiles. Given these immunologic similarities, perhaps the favorable response in AA with blockade of CXCR3 experimentally, and with ruxolitinib clinically, may be translated to T1DR.

Combination therapies Other possible therapeutic interventions include the use of a combination of thymoglobulin and GCSF which was reported effective in studies of patients diagnosed with T1D.49 A 2-year follow-up report50 demonstrated relatively preserved C-peptide secretion (area under the curve during MMTT) as well as immunologic markers suggesting an increase in peripheral blood levels of regulatory T cells (T regs). Similar findings were reported in the study with alefacept.29 In the most recent randomized, double-masked, placebo-controlled trial, the addition of GCSF did not enhance c-peptide preservation that was seen in the low dose (2.5 mg/kg) thymoglobulin group.51 However, in neither group was c-peptide preserved at 1 year, similar to the trial of higher dose (6.5 mg/kg) thymoglobulin at 2 years.52

Future considerations In the current iteration of therapies aimed at treatment for T1D, a combination approach appears to be rational. This combination could include a number of therapeutic agents, such as: (1) antiinflammatory drugs, (2) immunomodulation, (3) agents that favorably impact the development, recruitment or preservation of T regs, (4) diabetes-related antigen, and (5) those medications that may contribute to the preservation of beta cell health.53,54 Possible candidates for each category include: (1) Antiinflammatory agents, for example, antitumor necrosis factor (TNF) alpha, anti-IL1 beta, or anti-IL-6 agents.54 (2) The following immunomodulatory agents: thymoglobulin with or without GCSF,51,52 campath, anti-CD3, rituximab (anti-CD20), or abatacept (co-stimulation blockade) for an effect on the islet cell infiltrates present on pancreas or pancreas transplant biopsies. The availability of an anti-memory T cell agent would probably offer the best therapeutic advantage in this category. A possible option for the future may include dimethyl fumarate (DMF), currently used in psoriasis (Europe),55 and multiple sclerosis (MS) (the United States and Europe),56 which appears to inhibit memory T cell activity, along with targeting CXCR3+ T follicular cells and increasing T regs.56–58 Recent evidence suggests that DMF mechanism of action involves the downregulation of aerobic glycolysis,59 with a preferential effect on the pro-inflammatory Th1 and Th17 cells that depend on aerobic glycolysis for energy; whereas differentiation of T regs is driven by oxidative metabolism. An important safety concern for both MS and psoriasis involves the development of progressive multifocal leukoencephalopathy (PML), an opportunistic brain infection caused by the John Cunningham (JC) virus that has occurred in patients with either MS or psoriasis being treated with DMF. Although rare, it is a serious complication. However, PML seems to only occur in the context of severe lymphopenia, so that careful monitoring, and discontinuation of DMF in the context of significant lymphopenia (<500 cells/mm3) should minimize the risk.58,60 (3) The addition of agents that would increase the likelihood of enhancing a T reg response, for example, low-dose IL-2, GCSF (granulocyte colony stimulating factor) or T reg infusions, may also contribute to the reestablishment of tolerance necessary to restore the balance to a dysregulated immunologic landscape.53,54 (4) The use of diabetes-related antigen, for example, oral insulin or GAD vaccine.53,54 (5) Examples of beta cell protective agents that might further aid in the recovery of islet cells under the onslaught of the autoimmune attack of T1D might include: dipeptidyl

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Acknowledgment

peptidase 4 (DPP-4) inhibitors, glucagon-like peptide-1 receptor agonists (GLP-1 agonists), sodium-glucose cotransporter type1/2 (SGLT-1/2) inhibitors, biguanides (metformin), and/or insulin.53,54

Network for the pancreas organ donors with diabetes Network for the pancreas organ donors with diabetes (nPOD) is an organization inspired by the late George Eisenbarth that was established to obtain pancreases from human organ donors with T1D to support research on the pathogenesis of human disease.61 Since human ­pancreas tissue from organ donors would be preserved with Belzer’s (University of Wisconsin) preservation ­solution that is used routinely for flushing donor organs, the pancreas would be protected from autodigestion. Thus, this could be an important source of tissue for (1) identifying targets/biomarkers for potential therapeutic use in T1D, as well as (2) providing critical material relevant to dissecting autoimmune mechanisms involved in human T1D that cannot be addressed by studying NOD mice. Over the last decade, this project has expanded to include hundreds of donors, and increasing numbers of patients with T1D at various points of time related to T1D onset. Our work with T1DR has been complementary, and incorporated into the nPOD effort, where it is known as nPOD-T (nPOD-Transplantation).62 The native T1D and/or transplanted pancreas (T1DR) show many histological similarities, suggesting that the study of T1DR may shed light on the T1D process. Furthermore, the study of human pancreas tissue, by a number of laboratories around the world, with expertise in viruses, molecular biology, immunology, matrix proteins, and islet cell biology, is felt to be one of the more promising approaches to elucidating the immune-pathologic mechanisms of T1D.61,62 It should be noted that the insulitis seen in biopsies of our SPK recipients with T1DR generally involves approximately 30% of the total islet cell mass. This is similar to what was demonstrated in the pancreas of a 12-year-old girl with recently diagnosed T1D and diabetic ketoacidosis reported in 1985 by Gianfranco Bottazzo63 and has been supported by numerous studies,64 including studies of nPOD donors.65 This raises the question: if insulitis and beta cell loss are responsible for the hyperglycemia identified in both T1D and T1DR at the time of presentation, why is it that only a moderate proportion of the pancreas appears to be involved? This observation supports the concept that ongoing stress/inflammation is occurring in the pancreas or pancreas transplant, and may likely be related to the infiltrate but reflects more of an injury pattern than actual cellular death. It also provides more hope that this lesion may be reversible, since there appears to be a

significant proportion of viable islets. There is growing evidence that beta cell function is impaired in association with inflammation and stress in T1D and T1DR.66 Another addition to combination therapy might include results from nPOD studies demonstrating alterations in extracellular matrix components that may be amenable to therapeutic manipulation. For example, 4-methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis, and increased deposition of pro-inflammatory HA has been shown in the islets, pancreatic lymph nodes, and spleen of organ donors with T1D. Moreover, 4-MU therapy in the NOD mouse model prevented the development of diabetes,67 4-MU reduced HA accumulation, prevented islet cell damage by T cells, and increased FOXP3+ T regs in the islets.67 Since 4-MU is already available clinically to treat biliary spasm, it is one more potentially relevant agent in the combinatorial approach to successfully treat T1DR and, possibly, T1D. Finally, there is a growing sense that continuation of an immunosuppressive agent or combination therapy, rather than short-term treatment will be needed for resolution of the autoimmune process in T1D.54 This would be analogous to maintenance antirejection therapy in transplantation of solid organs or chronic medication necessary for treating psoriasis or other autoimmune disorders. Although our patients with T1DR have developed recurrent autoimmunity while taking chronic immunosuppressive medications, we are hopeful that innovative combination therapy and the use of selective agents, guided by our search and that of others in the field, in particular the nPOD team, for biomarkers, will lead to effective therapy.

Conclusions T1DR, which occurs in about 5% of SPKT recipients, 5–20 years after transplantation, appears to occur in association with autoantibody conversion, and the presence of autoreactive, memory CD4+ and CD8+ T cells. Pancreas transplant biopsies can provide critical information including the identification of membrane biomarkers on infiltrating T cells (CD2, CD45RO, CXCR3, etc.) that may lead to meaningful therapeutic intervention. The availability of new agents, for example, DMF, to target these biomarkers, particularly memory T cells, is crucial to the successful development of therapeutic approaches. The similarities between T1DR and T1D lead us to hope that effective treatment for T1DR may translate to successful therapy for T1D in the future.

Acknowledgment The authors would like to express their appreciation to Ms. Saggui Villalobos for her expert help in editing the manuscript, figures, and legends.

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24.  Recurrence of type 1 diabetes following simultaneous pancreas-kidney transplantation

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A.  Whole pancreas allo-transplantation



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A.  Whole pancreas allo-transplantation