American Journal of Transplantation C Copyright 2012 The American Society of Transplantation Wiley Periodicals Inc. and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2011.03977.x

Potent Induction Promotes Long-Term Insulin Independence After Islet Transplantation in Type1Diabetes

M. D. Bellina,*,F.B.Bartonb, A. Heitmanb,J. ITA, islet transplant alone; PII, potent induction im- Harmon,c A. N. Balamurugan,a R. Kandaswamy,c munosuppression; PTA, pancreas transplant alone; a d a SHE, severe hypoglycemic episodes; T1D, type 1 dia- D.E. Sutherland, R. Alejandro and B. J. Hering betes; TCDAb, T-cell depleting ; TNF-alpha-i, TN-alpha-inhibition; UMN, University of Minnesota. aThe Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN b Received 25 September 2011, revised 2 December 2011 The EMMES Corporation, Rockville, MD and accepted for publication 19 December 2011 cDepartment of Surgery, University of Minnesota, Minneapolis, MN dDiabetes Research Institute, University of Miami, Miami, FL ∗ Corresponding author: Melena Bellin, Introduction [email protected] mellitus (T1D) continues to present ther- The seemingly inexorable decline in insulin indepen- apeutic challenges. Intensive insulin therapy reduces the dence after islet transplant alone (ITA) has raised con- risk of microvascular complications but is limited by an in- cern about its clinical utility. We hypothesized that creased risk of life-impacting hypoglycemia (1). Some pa- induction therapy determines tients with long-standing T1D will develop unawareness durability of insulin independence. We analyzed the of hypoglycemia, preventing achievement of goal-glycemic proportion of insulin-independent patients following control. Successful intraportal allogeneic islet transplanta- final islet infusion in four groups of ITA recipients tion can substantially reduce glycemic variability, eliminate according to induction immunotherapy: University of severe hypoglycemia, reestablish hypoglycemia aware- Minnesota recipients given FcR nonbinding anti-CD3 alone or T-cell depleting antibodies (TCDAb) ness and permit insulin independence by restoring endoge- and TNF-a inhibition (TNF-a -i) (group 1; n = 29); recipi- nous insulin secretion (2–5). ents reported to the Collaborative Islet Transplant Reg- istry (CITR) given TCDAb+TNF-a -i (group 2; n = 20); A majority of islet transplant recipients at experienced cen- CITR recipients given TCDAb without TNF-a -i (group ters achieve insulin independence within the first year af- 3; n = 43); and CITR recipients given IL-2 receptor anti- ter transplant (2–4). Recipients are protected from severe bodies (IL-2RAb) alone (group 4; n = 177). Results were hypoglycemia, the most common indication for islet trans- compared with outcomes in pancreas transplant alone plant. However, early studies suggest a rapid loss of insulin (PTA) recipients reported to the Scientific Registry of independence beyond 1 year in many patients (2,3). Iden- Transplant Recipients (group 5; n = 677). The 5-year tifying transplant strategies which minimize the decline in insulin independence rates in group 1 (50%) and group 2 (50%) were comparable to outcomes in PTA (group 5: insulin independence is critical to developing islet trans- 52%; p>>0.05) but significantly higher than in group plant as an effective therapeutic intervention for T1D. 3(0%;p= 0.001) and group 4 (20%; p = 0.02). Induc- tion immunosuppression was significantly associated Proposed factors contributing to islet graft decline in- with 5-year insulin independence (p = 0.03), regardless clude alloimmune rejection, autoimmune recurrence (6), of maintenance immunosuppression or other factors. toxicity (7) and nonimmunologic These findings support potential for long-term insulin factors including exhaustion of a marginal beta-cell mass independence after ITA using potent induction therapy, (8). It is unclear whether manipulation of immune modu- + a with anti-CD3 Ab or TCDAb TNF- -i. lating therapies can prolong insulin independence. Most islet transplant recipients in the recent era received IL-2 Key words: , antithymocyte globulin, receptor antibodies (IL-2RAb; or ) CD3, islet transplant, OKT3, for induction (2). Within this population, insulin indepen- dence declines significantly after 1 year (2,3). Another ap- Abbreviations: ATG, anti-thymocyte globulin; CNI, proach to induction therapy involves the administration of -inhibitor; IL-2RAb, IL-2 receptor antibody; potent induction immunosuppression (PII) regimens, with Bellin et al. either FcR-nonbinding anti-CD3 or the munosuppression agents were coded as ever or never given and grouped novel combination of antithymocyte globulin (ATG) or alem- in categories according to mode of action, over all infusions for a re- tuzumab combined with tumor necrosis factor-alpha inhi- cipient. Maintenance immunosuppression, unless stated otherwise, con- bition (TNF-a-i) (9,10). These PII protocols target T cells, sisted of a low-dose calcineurin inhibitor (, cyclosporine) with while preserving or expanding regulatory T cells and their either mTOR inhibitor therapy (, ) or mycophenolate function, and minimizing cytokine toxicity on transplanted acid/mycophenolate mofetil. islets. Outcome measures The proportion of insulin-independent patients (defined by no exogenous The mechanism by which these induction immunosuppres- insulin use for ≥14 consecutive days) was determined at 1, 3 and 5 years sion regimens may exert benefit is twofold. First, PII may following the final islet infusion in groups 1–4. Rates of insulin indepen- provide better early posttransplant protection of islets from dence following PTA were obtained from UNOS/SRTR for pancreas graft innate and alloimmunity, thereby increasing the engrafted survival, equivalent to euglycemia without the need for exogenous in- beta-cell mass (11). Second, we speculate that these reg- sulin therapy (UNOS/SRTR 2009 Annual Report Table 1) (17). Fasting C- imens may mitigate recurrent autoimmunity directed at peptide to glucose ratios [calculated as C-peptide (ng/mL) × 100 divided transplant islets. This is supported by preclinical observa- by glucose (mg/dL)] were evaluated at 28 days posttransplant, to reflect tions that T-celldepletion therapy reverses diabetes in NOD functional beta-cell mass in the early posttransplant period. Autoantibody status at pretransplant baseline was defined by the following antibodies: mice (12,13), and clinical studies documenting prolonged glutamic acid decarboxylase antibody, insulin autoantibody and islet cell an- C-peptide production in type 1 diabetics receiving short- tibody. Other outcome measures included complete graft failure (C-peptide term monotherapy with anti-CD3 (14,15). <0.3 ng/mL) and the absence of severe hypoglycemic episodes (SHE) de- fined as requiring assistance by another person to restore euglycemia. In the present study, we investigate the impact of induction immunosuppression on long-term insulin independence in Statistical analysis islet transplant recipients at the University of Minnesota Baseline characteristics were compared across the four groups. Continuous (UMN) and in the Collaborative Islet Transplant Registry measures were compared by the Wilcoxon test and discrete measures by (CITR), which represents the largest collection of islet the chi-square test. transplant data available in the world to date. Those pa- tients receiving anti-CD3 monoclonal antibody alone, or Insulin independence and severe hypoglycemia at follow-up were analyzed ATG or alemtuzumab plus TNF-a-i exhibited greater long- as the percentage of patients meeting the endpoint annually post last infu- sion; missing data were considered missing at random (i.e. deducted from term insulin independence compared to patients treated both numerator and denominator). The data were analyzed by the general- with IL-2RAb.Outcomes are comparable to that seen with ized estimating equation (GEE) models with repeated measures within sub- pancreas transplant alone (PTA) (based on UNOS/SRTR ject to determine which factors were associated with insulin independence data). over time. Recipient, donor and islet characteristics, plus transplant cen- ter and immunosuppression categories (cumulative for all infusions) were first analyzed univariately. Factors significant at p < 0.10 were included in Materials and Methods multivariate models, and then stepped down at p < 0.05. Hazard ratios are reported for significant factors in final models. Transplant cohorts Islet allograft recipients meeting the following criteria were included in the Results analysis: (1) last islet infusion between 2002 and 2008, (2) at least 1 year of follow-up and (3) at least 50% of primary endpoint data reported (based Comparison of long-term insulin independence in ITA on November 2010 data lock). We analyzed outcomes in four groups of recipients treated with differing induction islet transplant alone ITA recipients according to induction immunotherapy: immunosuppression University of Minnesota recipients enrolled in single-center clinical trials (9,10,16) given FcR nonbinding anti-CD3 antibody teplizumab alone or the Patient, donor and islet graft characteristics are outlined in T-cell depleting antibody (TCDAb) ATG and TNF-a-i with , with Table 1. The islet transplant groups did not differ in terms or without daclizumab (group 1; n = 29); recipients reported to the Col- of baseline insulin use, weight or BMI. Total IEQ trans- laborative Islet Transplant Registry (CITR) given TCDAb + TNF-a-i (group 2; planted was slightly lower and islet autoantibodies were n = 20); CITR recipients given TCDAb without TNF-a-i (group 3; n = 43) more often positive in group 1. and CITR recipients given IL-2RAb alone (group 4, n = 177). Each individual allograft recipient was analyzed in only one of the four groups. The patients Observed rates of insulin independence in UMN patients from the University of Minnesota included in group 1 were excluded from receiving PII were 74% at 1 year, 50% at 3 years and 50% groups 2 or 3 (CITR groups). at 5 years. Forty percent were insulin independent at 10 years, although the number of cases that have yet reached Results were compared with outcomes in PTA recipients reported to the this time point is small (n = 2/5). Mean hemoglobin A1c Scientific Registry of Transplant Recipients (group 5; n = 677), obtained for the UNOS/SRTR 2002–2007 5-year cohort from the 2009 SRTR Annual levels were normal or near normal in insulin-independent Report (http://optn.transplant.hrsa.gov/ar2009/data_tables_section6.htm). recipients (5.5 ± 0.4% at 3 years, 5.6 ± 0.5% at 5 years).

ITA recipients received 1 to 6 donor infusions (1–3 in 98%). Selection of Insulin independence rates at 3 and 5 years post-last infu- immunosuppression was at the discretion of the transplant center. Im- sion (Figure 1) are superior for the groups 1 and 2 (UMN

American Journal of Transplantation doi: 10.1111/j.1600-6143.2011.03977.x Long-Term Insulin Independence After ITA

Ta b l e 1 : Baseline recipient characteristics, concomitant immunosuppression and transplant characteristics Group 2 CITR: Group 3 CITR: Group 4 Group 1 UMN TCD+TNF-a-i TCD-TNF-a-i CITR:IL2RAb PTA p2 N (%) or N (%) or N (%) or N (%) or N (%) or mean ± SE mean ± SE mean ± SE mean ± mean ± SE SE Era 29 22 41 177 1104 1999–2003 16 (55%) 3 (14%) 3 (7%) 86 (46%) 3413 <0.01 2004–2006 4 (14%) 11 (50%) 10 (24%) 78 (44%) 555 2007–2009 9 (31%) 8 (36%) 28 (68%) 18 (10%) 208 Patient characteristics Age at transplant (years) 40.6 ± 1.4 45.2 ± 1.5 44.3 ± 1.6 44.1 ± 0.8 33.3 ± 7.1 NS BMI (kg/m2) 23.6 ± 0.5 23.6 ± 0.5 23.2 ± 0.5 23.0 ± 0.2 – NS Weight (kg) 64.2 ± 1.7 63.6 ± 1.6 65.0 ± 1.6 64.9 ± 0.8 – NS Insulin use (u/day) 0.5 ± 0.01 0.6 ± 0.01 0.5 ± 0.01 0.5 ± 0.01 – NS Female 23 (79%) 14 (64%) 28 (68%) 104 (59%) 670 (60.7%) NS Presence of SHE 29 (100%) 17 (77%) 31 (76%) 137 (77%) - NS Immunosuppression, induction Antithymocyte globulin 18 (62%) 14 (64%) 36 (88%) .. 537 (48.6%) 1 Alemtuzumab - 6 (27%) 5 (12%) 267 (24.2%) Anti-CD3 mAb 11 (38%) 2 (9%) 36 (0.3%) IL-2 receptor antagonists 14 (48%) 10 (46%) 7 (17%) 177 164 (14.9%) (100%) TNF alpha inhibitors 15 (51%) 22 (100%) .. 29 (16%) – Other immunosuppression in first month posttransplant IMDPH () 3 (10%) 14 (64%) 34 (83%) 28 (16%) (58.6%) <0.01 m-TOR inhibitors 28 (97%) 17 (77%) 26 (63%) 173 (98%) (16.5%) 0.03 Calcineurin inhibitors 25 (86%) 22 (100%) 21 (51%) 176 (99%) (91.6%) 0.04 3 (10%) 5 (12%) 12 (7%) NS Infusions 1 21 (72%) 8 (36%) 14 (34%) 36 (20%) – <0.01 2 7 (24%) 8 (36%) 18 (44%) 81 (46%) – 3 1 (3%) 4 (18%) 8 (20%) 59 (33%) – ≥4 .. 2 (9%) 1 (2%) 1 (0.6%) – ∗ Cumulative IEQs ( 100 000) 614 ± 46 889 ± 88 908 ± 87 906 ± 32 – 0.01 Donor/graft characteristics Donor BMI 34 ± 1.5 31.1 ± 1.1 29.7 ± 0.9 29.0 ± 0.4 – 0.003 Donor age 32 ± 3 45.1 ± 2.1 45 ± 343± 1–<0.001 Cold ischemia time 6.0 ± 0.5 6.8 ± 0.4 6.8 ± 0.4 7.5 ± 0.2 12.2 ± 3.5 NS Packed cell volume 5.0 ± 0.8 7.7 ± 0.9 7.7 ± 0.9 8.4 ± 0.4 – 0.02 ∗ The 2009 UNOS/SRTR report did not include any transplants done in 2009. SHE = severe hypoglycemic episodes; IMDPH = ionosine monophosphate dehydrogenase, including mycophenolic acid and mycophe- nolate mofetil; m-TOR = mammalian target of rapamycin and includes sirolimus and everolimus; TNF-alpha = tumor necrosis factor-alpha. TNF-alpha inhibitors include etanercept and infliximab. Calcineurin inhibitors include tacrolimus and cyclosporine. T-cell depleting agents include anti-CD3 monoclonal antibodies, antithymocyte globulin, alemtuzumab. 1These define the study categories, hence testing is not necessary. 2p-values relate to differences among the islet transplant groups (do not include the PTA). 3Results are shown here and in Figure 1 for PTAs done in 2002–2007, comprising the 5-year cohort of US Scientific. Registry of Transplant Recipients, thus, PTAs transplanted in 1999–2001 are not included. and TCDAb+TNF-a-i) compared to group 3 (TCDAb with- Insulin independence rates were significantly higher when out TNF-a-i) (p ≤ 0.01 at 3 years and 0.001 at 5 years) and TNF-a-i was given in the peritransplant period. TNF-a-i con- group 4 (IL2R-Ab recipients) (p = 0.05 at 3 years and 0.002 ferred substantial benefit with ATG or alemtuzumab induc- at 5 years), a difference which was sustained within the tion (group 2), and marginal benefit with daclizumab (group subset of autoantibody positive patients. Observed rates 4) (Figure 2). TNF-a-i with anti-CD3 monoclonal antibody of insulin independence in groups 1 and 2 were 50% and was never used. 45% respectively at 3 years and 50% in both groups at 5 years. The proportion of insulin independence at 3 and Virtually everyone in group 2 (TCDAb+TNF-a-i) received 5 years in these two groups was comparable to that calcineurin inhibition (CNI), while only about half of group of PTA (group 5), based on registry data from SRTR 3 (TCDAb without TNF-a-i) received CNI. However, when (p >> 0.05). subdividing group 3 by the presence or absence of CNI,

American Journal of Transplantation doi: 10.1111/j.1600-6143.2011.03977.x Bellin et al.

Figure 1: Observed insulin independence in pancreas transplant alone and in islet trans- plant alone recipients based on induction im- munosuppression administered. Islet transplant groups are indicated as follows: group 1 (UMN re- cipients, solid black), group 2 (CITR recipients re- ceiving TCDAb +TNF-a-i, solid gray), group 3 (CITR recipients receiving TCDAb without TNF-a-i, gray checkered), group 4 (CITR recipients receiving IL- 2RAb, vertical lines), group 5 (pancreas transplant alone recipients, white). Insulin independence was superior in groups 1 and 2 compared to groups 3 and 4 at 3 and 5 years posttransplant; groups 1 and 2 did not differ statistically from pancreas ∗ ∗∗ transplant alone (group 5). p<0.01; p≤0.05. both CNI +/− groups experience substantial graft function Reduction in severe hypoglycemia posttransplant losses over 5 years relative to group 2. Outcomes were Severe hypoglycemic episodes were uncommon in all ITA poor in group 3, regardless of CNI use—no patient in the recipients, regardless of induction therapy. Over three- CNI +subgroup and fewer than 10% of patients in the quarters of ITA recipients suffered from severe hypo- CNI—subgroup were insulin independent at 3 years, and glycemic episodes prefirst transplant. In contrast, over none were insulin independent at 5 years. Hence, there is 80% of ITA recipients in all groups at 1–3 years posttrans- no evidence that the superior outcome in group 2 is due plant reported the absence of severe hypoglycemia. to the confounding effect of CNI. Adjusted analysis of effect of TCDAb and TNF-a -i on Comparison of early posttransplant islet graft insulin independence function, measured by fasting C-peptide to glucose An adjusted analysis was performed to determine the ratios relative impact of induction immunosuppression when The fasting C-peptide to glucose ratio at day 28 posttrans- accounting for potential confounding variables, includ- plant was higher in group 1 (1.2 ± 0.1) compared to group ing other immunosuppression and baseline characteristics 3(0.6± 0.1) and group 4 (0.8 ± 0.1; p ≤ 0.001). There was (Table 1). Insulin independence decreased over time in all a trend toward higher C-peptide to glucose ratios in group groups. TCDAb or anti-CD3 (p = 0.03), TNF-a-i (p = 0.03), 2(0.9± 0.1) compared to group 3 (p = 0.12) and group and length of follow-up (p < 0.0001) emerged as significant 4(p= 0.23). Long-term retention of any graft function (C- variables associated with insulin independence regardless peptide production ≥0.3 ng/mL) was greater when TNF-a-i of other recipient, donor, graft or immunosuppression vari- was administered with TCDAbs (data not shown). ables. None of the donor, islet or recipient preinfusion

Figure 2: Insulin independence by induction agent, in the presence of TNF-alpha inhibitor therapy (wide bars) or absence of TNF-alpha in- hibitor (narrow bars). Insulin indepen- dence was significantly lower at every time point in the ATG group in the ab- sence of TNF-alpha inhibitor as com- pared to ATG with TNF-alpha inhibitor.

American Journal of Transplantation doi: 10.1111/j.1600-6143.2011.03977.x Long-Term Insulin Independence After ITA characteristics included in the model blunted or explained Early studies in alloislet transplant documented sustained the effects of T-cell depletion or TNF-a−i on insulin inde- insulin independence primarily in the context of multiple pendence. donor infusions to increase transplanted beta-cell mass (3,5,25). Induction immunosuppression regimens that in- hibit innate and adaptive immunity protect islets from immune-mediated destruction early posttransplant and Discussion thereby increase engrafted beta-cell mass, insulin secre- tory capacity, and islet reserve. Single-center trials have The decline in insulin independence rates beyond 1 year af- documented achievement of insulin independence with ter alloislet transplant for T1D has raised concern about the fewer islets and sustained insulin independence with a therapeutic value of islet allografts. Proposed factors con- single islet donor when induction comprised ant-CD3 or a tributing to islet loss include nonimmunologic injury such TCDAb and TNF- -I (9,10,26). Insulin secretory capacity as amyloid-mediated islet apoptosis (18) and immunologic in response to glucose-potentiated arginine (a surrogate factors such as recurrent beta-cell autoimmunity (6,19). marker of beta-cell mass) is superior in patients receiving + a We demonstrate here that patients receiving potent induc- TCDAb TNF- -i compared to IL-2RAb induction immuno- tion immunosuppression, with FcR nonbinding anti-CD3 suppression (11). or either ATG or alemtuzumab with TNF-a inhibitors, are more than twice as likely to maintain long-term insulin We postulate that these PII regimens may have further independence for ≥5 years posttransplant compared to benefit through their effect on the autoimmune process those receiving IL-2 receptor antagonists, despite the fact of T1D. Autoimmunity has been identified as one cause that only a single donor pancreas was utilized in over 70% of pancreas transplant failure, as evidenced by autoreac- of group 1 recipients. Such regimens may benefit long- tive T cells and insulitis in pancreas allografts (27). In an- term outcomes through improved engrafted islet mass and imal models, islets are even more susceptible to recur- minimization of recurrent autoimmunity. Insulin indepen- rent autoimmunity (28,29). In clinical islet transplant recipi- b dence rates in these recipients approach those seen in ents, the presence of T-cellautoreactivity to -cell PTA. These data demonstrate the potential for sustained is associated with failure to achieve or delayed achieve- insulin independence after alloislet transplantation for T1D ment of insulin independence (19). Whole organ are much and highlight the importance in selection of induction im- less prone to recurrent autoimmune destruction than islet munosuppression. grafts in immunosuppressed recipients, presumably due to immunomodulatory effects of donor T cells cotransplanted Initial reports evaluating long-term insulin independence in with peripancreatic lymph nodes (29). Thus, islet transplant islet allotransplant recipients receiving IL-2RAb suggested protocols must adequately protect from recurrent autoim- frequent return to insulin dependence after 1 year post- munity for optimal success. transplant, with ∼10% of recipients insulin independent at 5 years (2). The present analysis shows half of patients Anti-CD3, ATG and alemtuzumab may manifest a long-term treated with PII maintained insulin independence through benefit through restored self-tolerance. In clinical trials for 5 years, a proportion comparable to that seen in PTA new onset T1D, short-term monotherapy with anti-CD3 recipients. monoclonal antibody has resulted in sustained superior C- peptide production and lower insulin requirements (14,15). Although much research has focused on the immunologic In contrast, no such benefit was observed in recent on- factors contributing to islet loss, nonimmunologic factors, set T1D patients receiving the IL-2RAb daclizumab plus in particular the exhaustion and decline of a marginal mass mycophenolate mofetil (30). In pancreas transplant recip- b likely play a role (20). Constant overstimulation of an in- ients, although daclizumab suppressed -cell autoimmu- adequately low islet mass causes endoplasmic reticulum nity while sparing T-cell memory responses to bacterial stress-mediated islet apoptosis (21,22), a potential media- and viral antigens, the effect was short-lived, limited to the tor of gradual islet loss. This is supported by recent obser- duration of drug action; in contrast, ATG resulted in pro- > vations of wide spread amyloid deposition in transplanted longed alteration of the cellular autoreactivity, for 1year human islets (18), as well as observations of gradual loss after drug administration (31). of insulin independence in islet autotransplants (which are not subject to immune destruction) in canines (23) and in The mechanism for prolonged abrogation of autoimmunity humans with a marginal islet mass (<2500 IEQ/kg) (24). likely relates to the potential of these agents to enhance Under this reasoning, the transplanted and engrafted islet T regulatory cells (Tregs) and shift the balance of Tregs mass in IL-2RAb-treated patients was borderline adequate and memory T-effector cells toward a more tolerant phe- to facilitate short-term insulin independence but insuffi- notype (13,32–36). Treatment with anti-CD3 mAb or an- cient to sustain long-term graft survival. In contrast, we tilymphocyte serum in NOD mice restores self-tolerance hypothesize that sustained islet graft survival is achievable to beta-cell antigens and results in prolonged reversal of in patients receiving PII, through engraftment of a higher diabetes (12,13). Both agents can induce expansion of + + + proportion of transplanted islets. foxp3 CD4 CD25 and, in the case of anti-CD3, also

American Journal of Transplantation doi: 10.1111/j.1600-6143.2011.03977.x Bellin et al.

CD8+CD25+ T-regs (13,32–34). This effect of ATG is pre- particularly with regard to insulin independence, and pro- served or even enhanced in the presence of tacrolimus vide the best opportunity for comparison of these two (35). Islet transplant recipients treated with alemtuzumab groups outside a clinical trial. Notably, this analysis was had an increase in CD4+foxp3+Tregs that was not seen restricted to islet transplant alone versus solitary pancreas with daclizumab (36). transplant (without kidney transplant) for this analysis, in order to maintain comparability. Coadministration of TNF-a-i at induction resulted in a greater likelihood of insulin independence in the multivari- In conclusion, these results highlight the potential for pro- ate model, particularly when ATG or alemtuzumab were longed insulin independence after alloislet transplant when used for induction. TNF-a-i may mediate this benefit by potent induction immunosuppression is used, and empha- two proposed mechanisms. First, TNF-a-i may prevent the size the critical impact of induction immunosuppression on detrimental effects of cytokine exposure at the time of islet long-term islet graft function. Prolonged insulin indepen- infusion. TNF-a exposure is known to be cytotoxic to hu- dence was superior in alloislet transplant recipients receiv- man islets in culture (37), and innate immunity—including ing PII, with either FcR nonbinding anti-CD3 monoclonal cytokine toxicity—in the peritransplant period is one pro- antibody alone, or ATG or alemtuzumab plus TNF-a-i for posed contributor to islet loss (38). In heart allograft mod- induction immunosuppression compared to IL-2RAb in- els, TNF-a-i substantially reduces early leukocyte infiltration duction. This benefit may in part be mediated by im- of the allograft and prolongs graft survival (39). The protec- proved islet engraftment and mitigation of autoreactive tive effect of TNF-a-i may be more important with an agent T-cell responses. These results show that with potent in- such as ATG, which is well-known to induce a cytokine re- duction strategies, intraportal alloislet grafts can provide lease syndrome in the recipient (40). Second, TNF-a-i may prolonged glycemic benefits in selected recipients, ap- have a sustained impact on autoimmunity. The timing of proaching those seen with solitary vascularized pancreas TNF-a-I exposure is likely critical. TNF-a-i increases Tregs grafts, with a far less invasive impact. and completely prevents onset of autoimmune diabetes in NOD mice only if given in the neonatal period but not when administered later (41,42). Similarly, TNF-a-i may be particularly effective when given peritransplant (i.e. during Acknowledgments the initial period of exposure). The authors are indebted to the Investigators of the Collaborative Islet Trans- plant Registry (CITR) for their contribution of data and critical appraisal of This is the largest study to date reporting such a sus- the manuscript. The CITR is funded by the National Institute of Diabetes tained benefit of induction therapy in islet transplantation. and Digestive and Kidney Diseases (NIDDK) with supplemental funding by One limitation is that results derive from a voluntary, al- the Juvenile Diabetes Research Foundation. Dr. Melena Bellin is supported though audited, registry rather than a controlled trial. In by a K23 grant from the NIDDK (1K23DK084315-01A1). The University of order to account for this limitation, we controlled for po- Minnesota islet transplant program has been supported in part by grants tential confounders in a multivariate analysis that included from the National Institutes of Health, National Institute of Allergy and patient and islet graft characteristics, concurrent mainte- Infectious Diseases (5U01 AI-065193), National Institute for Diabetes, Di- nance immunosuppression and transplant center. We ac- gestive and Kidney Diseases (DK56963), the National Center for Research knowledge that there are some limitations inherent in the Resources (MO1-RR00400 and U42 RR016598-01) and the Juvenile Dia- betes Research Foundation (JDRF #4-1999-841 and 4-2008-386). The data registry despite the controlled analysis. Patients were cat- and analyses reported in the 2009 Annual Report of the US Organ Procure- egorized as ever treated with a particular immunosuppres- ment and Transplantation Network and the Scientific Registry of Transplant sion agent over time, and were not able to account for Recipients have been supplied by UNOS and Arbor Research under contract changes in maintenance immunosuppression and/or ad- with HHS/HRSA. The authors alone are responsible for reporting and inter- equacy of immunosuppression (trough levels). The num- preting these data; the views expressed herein are those of the authors ber of patients, although large for the islet transplant field, and not necessarily those of the US Government. is small within the subgroups and diminishes over time. In addition, mechanistic data, including cellular autoimmu- nity assays are not available for these recipients. Although Disclosure autoantibody data are contained within the registry, au- toantibodies are poor markers for the presence of active The authors of this manuscript have no conflicts of inter- autoimmunity (19). est to disclose as described by the American Journal of Transplantation The comparison between the CITR islet transplant and the UNOS/SRTR whole pancreas alone data is limited by the References use of two separate data registries, and results must be carefully interpreted within this context. While not random- 1. Cryer PE. The barrier of hypoglycemia in diabetes. Diabetes 2008; ized, this comparison is concurrent and based on the most 57: 3169–3176. complete data available. Both CITR and UNOS/SRTR col- 2. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical lect similar data in transplanted type 1 diabetic recipients, islet transplantation. Diabetes 2005; 54: 2060–2069.

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3. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the 22. Bonora E. Protection of pancreatic beta-cells: Is it feasible? Nutr Edmonton protocol for islet transplantation. N Engl J Med 2006; Metab Cardiovasc Dis 2008; 18: 74–83. 355: 1318–1330. 23. Alejandro R, Cutfield RG, Shienvold FL, et al. Natural history of 4. Robertson RP. Islet transplantation a decade later and strategies intrahepatic canine islet cell autografts. J Clin Invest 1986; 78: for filling a half-full glass. Diabetes 2010; 59: 1285–1291. 1339–1348. 5. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in 24. Sutherland DE, Radosevich D, Bland B, et al. Total pancreatectomy seven patients with type 1 diabetes mellitus using a glucocorticoid- and islet autotransplant for chronic pancreatitis: Early outcomes in free immunosuppressive regimen. N Engl J Med 2000; 343: recent cases. [Abstract]. Pancreas 2009; 38: 1051. 230–238. 25. Warnock GL, Kneteman NM, Ryan E, Seelis RE, Rabinovitch A, Ra- 6. Monti P, Scirpoli M, Maffi P, et al. Islet transplantation in patients jotte RV. Normoglycaemia after transplantation of freshly isolated with autoimmune diabetes induces homeostatic cytokines that ex- and cryopreserved pancreatic islets in type 1 (insulin-dependent) pand autoreactive memory T cells. J Clin Invest 2008; 118: 1806– diabetes mellitus. Diabetologia 1991; 34: 55–58. 1814. 26. Gangemi A, Salehi P, Hatipoglu B, et al. Islet transplantation for 7. Nir T, Melton DA, Dor Y. Recovery from diabetes in mice by beta brittle type 1 diabetes: The UIC protocol. Am J Transplant 2008; 8: cell regeneration. J Clin Invest 2007; 117: 2553–2561. 1250–1261. 8. Keymeulen B, Korbutt G, De Paepe M, Gorus F, Kloppel G, 27. Vendrame F, Pileggi A, Laughlin E, et al. Recurrence of type 1 Pipeleers DG. Long-term metabolic control by rat islet grafts de- diabetes after simultaneous pancreas–kidney transplantation, de- pends on the composition of the implant. Diabetes 1996; 45: spite immunosuppression, is associated with autoantibodies and 1814–1821. pathogenic autoreactive CD4 T-cells. Diabetes 2010; 59: 947– 9. Hering BJ, Kandaswamy R, Harmon JV, et al. Transplantation of 957. cultured islets from two-layer preserved pancreases in type 1 dia- 28. Naji A, Silvers WK, Bellgrau D, Barker CF.Spontaneous diabetes in betes with anti-CD3 antibody. Am J Transplant 2004; 4: 390–401. rats: Destruction of islets is prevented by immunological tolerance. 10. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, Science 1981; 213: 1390–1392. marginal-dose islet transplantation in patients with type 1 dia- 29. Bartlett ST, Chin T, Dirden B, Quereshi A, Hadley G. Inclusion of betes. JAMA 2005; 293: 830–835. peripancreatic lymph node cells prevents recurrent autoimmune 11. Rickels MR, Chiou AJ, Fuller C, et al. Beta-cell secretory capacity destruction of islet transplants: Evidence of donor chimerism. after human islet transplantation by the CIT07 versus Edmonton Surgery 1995; 118: 392–397; Discussion 397–398. protocol: Preliminary results. Am J Transplant 2011; 11(Suppl 2): 30. Gottlieb PA, Quinlan S, Krause-Steinrauf H, et al. Failure to pre- 79–80. serve beta-cell function with mycophenolate mofetil and da- 12. Maki T, Ichikawa T, Blanco R, Porter J. Long-term abrogation of clizumab combined therapy in patients with new-onset type 1 autoimmune diabetes in nonobese diabetic mice by immunother- diabetes. Diabetes Care 2010; 33: 826–832. apy with anti-lymphocyte serum. Proc Natl Acad Sci USA 1992; 31. van de Linde P, Vd Boog PJ, Tysma OM, et al. Selective unre- 89: 3434–3438. sponsiveness to beta cell autoantigens after induction immuno- 13. Chatenoud L. Immune therapy for type 1 diabetes mellitus—what suppression in pancreas transplantation with anti--2 re- is unique about anti-CD3 antibodies? Nat Rev Endocrinol 2010; 6: ceptor antibody versus anti-thymocyte globulin. Clin Exp Immunol 149–157. 2007; 149: 56–62. 14. Keymeulen B, Vandemeulebroucke E, Ziegler AG, et al. Insulin 32. Bisikirska B, Colgan J, Luban J, Bluestone JA, Herold KC. TCR needs after CD3-antibody therapy in new-onset type 1 diabetes. stimulation with modified anti-CD3 mAb expands CD8+ Tcell N Engl J Med 2005; 352: 2598–2608. population and induces CD8+CD25+ Tregs. J Clin Invest 2005; 15. Herold KC, Gitelman S, Greenbaum C, et al. Treatment of patients 115: 2904–2913. with new onset type 1 diabetes with a single course of anti-CD3 33. Lopez M, Clarkson MR, Albin M, Sayegh MH, Najafian N. A novel mAb Teplizumab preserves insulin production for up to 5 years. mechanism of action for anti-thymocyte globulin: Induction of Clin Immunol 2009; 132: 166–173. CD4+CD25+Foxp3+ regulatory T cells. J Am Soc Nephrol 2006; 16. Bellin MD, Kandaswamy R, Parkey J, et al. Prolonged insulin in- 17: 2844–2853. dependence after islet allotransplants in recipients with type 1 34. Gurkan S, Luan Y, Dhillon N, et al. Immune reconstitution following diabetes. Am J Transplant 2008; 8: 2463–2470. rabbit antithymocyte globulin. Am J Transplant 2010; 10: 2132– 17. 2010 Annual Data Report of the U.S. Organ Procurement and 2141. Transplantation Network and the Scientific Registry of Transplant 35. Sewgobind VD, van der Laan LJ, Kho MM, et al. The calcineurin Recipients. Department of Health and Human Services, Health inhibitor tacrolimus allows the induction of functional CD4CD25 Resources and Services Administration, Healthcare Systems Bu- regulatory T cells by rabbit anti-thymocyte globulins. Clin Exp Im- reau, Division of Transplantation, Rockville, MD. munol 2010; 161: 364–377. 18. Westermark GT, Westermark P, Berne C, Korsgren O, Nordic Net- 36. Toso C, Edgar R, Pawlick R, et al. Effect of different induction work for Clinical Islet Transplantation. Widespread amyloid depo- strategies on effector, regulatory and memory lymphocyte sub- sition in transplanted human pancreatic islets. N Engl J Med 2008; populations in clinical islet transplantation. Transpl Int 2009; 22: 359: 977–979. 182–191. 19. Huurman VA, Hilbrands R, Pinkse GG, et al. Cellular islet autoim- 37. Rabinovitch A, Sumoski W, Rajotte RV, Warnock GL. Cytotoxic munity associates with clinical outcome of islet cell transplanta- effects of cytokines on human pancreatic islet cells in monolayer tion. PLoS One 2008; 3: e2435. culture. J Clin Endocrinol Metab 1990; 71: 152–156. 20. Smith RN, Kent SC, Nagle J, et al. Pathology of an islet transplant 2 38. Lai Y, Chen C, Linn T. Innate immunity and heat shock response years after transplantation: Evidence for a nonimmunological loss. in islet transplantation. Clin Exp Immunol 2009; 157: 1–8. Transplantation 2008; 86: 54–62. 39. Ishii D, Schenk AD, Baba S, Fairchild RL. Role of TNFalpha in 21. Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin early chemokine production and leukocyte infiltration into heart Invest 2006; 116: 1802–1812. allografts. Am J Transplant 2010; 10: 59–68.

American Journal of Transplantation doi: 10.1111/j.1600-6143.2011.03977.x Bellin et al.

40. Hardinger KL. Rabbit antithymocyte globulin induction therapy early development of autoimmunity and the diabetogenic process. in adult renal transplantation. Pharmacotherapy 2006; 26: 1771– J Exp Med 1994; 180: 995–1004. 1783. 42. Wu AJ, Hua H, Munson SH, McDevitt HO. Tumor necrosis factor- 41. Yang XD, Tisch R, Singer SM, et al. Effect of tumor necrosis factor alpha regulation of CD4+CD25+ T cell levels in NOD mice. Proc alpha on insulin-dependent diabetes mellitus in NOD mice: I. The Natl Acad Sci USA 2002; 99: 12287–12292.

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