Long-Term Insulin-Independence After Allogeneic Islet Transplantation for Type 1 Diabetes: Over the 10-Year Mark

Results of islet of Langerhans transplantation have markedly improved in recent years, but most patients still lose insulin independence in the long-term. We report herein the longest (over 11 years) case of insulin independence after allogeneic islet transplantation. The subject had a 27-year history of type 1 diabetes and received a single islet-after-kidney graft of 8800 islet equivalents (IEQ)/kg, pooled from 2 donors. Insulin was discontinued by 3 months posttransplant and the patient has remained off insulin ever since. Yearly follow-up studies have revealed normal metabolic control, including normal oral glucose tolerance test (OGTT). Reasons for success may involve choice of immunosuppression, low metabolic demand and low immune responsiveness as suggested by an excellent HLA matching and a high count of circulating regulatory T cells. This observation is so far an exceptional case, but clearly demonstrates the validity of the concept that long-term insulin independence after allogeneic islet transplantation is an achievable target. Results of islet of Langerhans transplantation have markedly improved in recent years, but most patients still lose insulin independence in the long-term. We report herein the longest (over 11 years) case of insulin independence after allogeneic islet transplantation. The subject had a 27-year history of type 1 diabetes and received a single islet-after-kidney graft of 8800 islet equivalents (IEQ)/kg, pooled from 2 donors. Insulin was discontinued by 3 months posttransplant and the patient has remained off insulin ever since. Yearly follow-up studies have revealed normal metabolic control, including normal oral glucose tolerance test (OGTT). Reasons for success may involve choice of immunosuppression, low metabolic demand and low immune responsiveness as suggested by an excellent HLA matching and a high count of circulating regulatory T cells. This observation is so far an exceptional case, but clearly demonstrates the validity of the concept that long-term insulin independence after allogeneic islet transplantation is an achievable target. The application of the Edmonton protocol (1Shapiro AM Lakey JR Ryan EA et al.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen..N Engl J Med. 2000; 343: 230-238Crossref PubMed Scopus (4429) Google Scholar) has led to a spectacular improvement in the results of islet transplantation as a therapeutic approach to type 1 diabetes (2Marzorati S Pileggi A Ricordi C Allogeneic islet transplantation..Expert Opin Biol Ther. 2007; 7: 1627-1645Crossref PubMed Scopus (36) Google Scholar). Insulin independence rates at 1 year posttransplant are close to matching those of whole pancreas transplantation, but tend to decrease over time to a sobering 10–15% at 5 years (3Ryan EA Paty BW Senior PA et al.Five-year follow-up after clinical islet transplantation..Diabetes. 2005; 54: 2060-2069Crossref PubMed Scopus (1382) Google Scholar). Reasons for this progressive drop in functional capacity are numerous and may include acute and chronic immune rejection, recurrence of autoimmunity, immunosuppressive drug toxicity and failure of beta cells to regenerate (4Shapiro AMJ Lakey JR Paty BW et al.Strategic opportunities in clinical islet transplantation..Transplantation. 2005; 79: 1304-1307Crossref PubMed Scopus (123) Google Scholar). However, the main reason for graft function decline might simply be ‘exhaustion’ of the islet graft, in the context of marginal engraftment. This is emphasized by the demonstration that the metabolic response of the graft in glucose tolerance or arginine stimulation tests is most of the time abnormal, even in patients who are off-insulin with adequate HbA1c (5Ryan EA Lakey JRT Rajotte RV et al.Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol..Diabetes. 2001; 50: 710-719Crossref PubMed Scopus (787) Google Scholar.6Rickels MR Schutta MH Markmann JF et al.Beta-cell function following human islet transplantation for type 1 diabetes..Diabetes. 2005; 54: 100-106Crossref PubMed Scopus (97) Google Scholar). With this picture of the current status of islet transplantation, the analysis of cases of long-term insulin independence could be critical for the understanding of the determinants of long-term failure or success. We report herein the first case of islet allogeneic transplantation in a patient with type 1 diabetes to ever reach and pass the symbolic threshold of 10 years of insulin independence, with perfect metabolic control and discuss the unique features that may have been critical determinants of success. A 36-year old female patient with a 27-year history of type 1 diabetes received an islet transplant on June 21, 1996. She had been a recipient of two previous kidney transplants for end-stage diabetic nephropathy. The first kidney transplant performed in 1989 was rapidly lost due to arterial thrombosis and she was retransplanted in 1990. Her immunosuppressive regimen comprised an association of Ciclosporin A microemulsion (CsA), azathioprine (Aza) and prednisone. In the first year of the second kidney transplant, she experienced rejection episodes that responded to steroid boluses. At the time of islet transplant, her kidney function was decreased, with an isotopic glomerular filtration rate measured at 31.5 mL/min, but had been stable for the past 5 years. She was on a mean insulin dose of 16 units/ day, with a poor metabolic control (HbA1c at 11.2%), hypoglycaemia unawareness and extreme glycemic lability, for which she experienced several hypoglycaemic coma episodes each year. She tested positive for anti-GAD65 autoantibodies, but negative for anti-IA2. She had low but measurable basal C-peptide levels (190 pmol/L), without response to glucagon stimulation (140 pmol/L). These values fall close to the limits or outside the calibration range of the assay (166–2347 pmol/L; Immulite 2500, Siemens Healthcare Diagnostics, Deerfield, IL) and are therefore of questionable accuracy. Metabolic lability was the definite indication for performing the islet-after-kidney (IAK) transplant. At the time of transplantation, she had severe peripheral and autonomous neuropathy, with hypertension and ortho-statism and severe retinopathy, with a history of panretinal photocoagulation, left eye blindness and 60% visual acuity of the right eye. She had no evidence of macroangiopathy. She received an islet preparation of 528000 islet equivalents (IEQ) pooled from the pancreas of two deceased donors (106 700 and 421 300 IEQ from each donor), corresponding to 8800 IEQ/kg body weight. Islets were isolated using the automated Ricordi method with some local modifications (7Ricordi C Lacy PE Finke EH Olack BJ Scharp DW et al.Automated method for isolation of human pancreatic islets..Diabetes. 1988; 37: 413-420Crossref PubMed Scopus (1174) Google Scholar,8Bucher P Mathe Z Morel P et al.Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation..Transplantation. 2005; 79: 91-97Crossref PubMed Scopus (104) Google Scholar). The enzyme used for pancreas digestion was crude collagenase (Collagenase Type P, Boehringer-Mannheim, Germany), and islets were purified in discontinuous Ficoll density gradients in a COBE 2991 cell processor (COBE, Lakewood, CO). Islets were conditioned into 50-mL syringes and transplanted intraportally under local anesthesia by a transhepatic percutaneous approach. A 3000-unit heparin bolus was injected intraportally immediately prior to islet infusion. The procedure was uneventful. She received immunosuppressive induction with equine antithymocyte globulin (ATGAM, Pfizer, New York, NY) at 10 mg/kg/day for 10 days. Intravenous (iv) methylprednisolone boluses (500 mg on day 1, 100 mg on day 2) were administered before the first ATGAM infusions, and steroids were then rapidly tapered to 5 mg prednisone daily over 3 months. Maintenance immunosuppression was achieved with CsA aiming for target trough levels of 100–150 ng/mL, and Aza at 1 mg/kg/day. Aza was switched to mycophenolate mofetil at 250 mg bid one year after transplant. Intensive insulin therapy was administered for 2 weeks after transplant via an iv pump, in order to maintain blood glucose levels within the normal range. Patient was discharged after 2 weeks on subcutaneous insulin and followed up thereafter as an outpatient. By 1-month posttransplantation, insulin could be reduced by half to 8 units/day and by 3 months, on September 22, 1996, insulin was discontinued altogether. At the time she was taken off insulin, she had basal and stimulated (glucagon-stimulation test) C-peptide levels of 680 and 1500 pmol/L, respectively and her HbA1c had normalized to 4.8%. Since that day, these values have always been within the normal range and are recapitulated in Figure 1. In particular, her HbA1c has never been higher than 5.8%. She underwent yearly insulin stimulation tests by intravenous (IVGTT) and oral (OGTT) glucose tolerance tests (9Berney T Toso C et al.Monitoring of the islet graft..Diabetes Metab. 2006; 32: 502-512Crossref Google Scholar). Her IVGTT showed glucose disposal rates (KG) varying over the years between −1.2 and −3.5 (normal < −1.0) and acute insulin responses varying between 20.4 mU/L and 63.7 mU/L (normal > 20 mU/L). Her OGTT showed 2-h postload blood glucose values varying between 5.0 mmol/L and 9.4 mmol/L; she met the criteria for impaired glucose tolerance (blood glucose 7.9–11.0 mmol/L) on only 2 occasions, all other tests fell within the definition of normal glucose tolerance (blood glucose ≥7.8 mmol/L; 10). Interestingly, her kidney function has been relatively stable over the years, with isotopic glomerular filtration rates measured annually varying between 30.9 and 41.8 mL/min. With respect to other microangiopathy complications, peripheral and autonomous neuropathy and retinopathy, already severe at the time of transplantation, have remained stable. She underwent bilateral cataract surgery 2 and 4 years after islet transplantation. In terms of macroangiopathy, she has remained asymptomatic for cardiomyopathy and a thallium-dipyridamole stress myocardial scintigraphy performed 11 years after transplantation was normal. She experienced 2 episodes of hemorrhagic stroke, 7 and 11 years posttransplant. In both instances, stroke was secondary to bouts of hypertension, led to intrathalamic hematoma and resolved rapidly and without sequelae. On the immunologic standpoint, the patient received two kidneys and an islet preparation pooled from 2 donors. There were multiple HLA mismatches (MM), but no HLA repeat MM between the islets and the two kidneys or between the 2 islet donors (Table 1). The determination of anti-HLA antibody was performed over time by complement-dependant cytotoxicity (CDC) to determine the PRA (%) and by solid phase assay (ELISA and Luminex single antigen). De novo anti-HLA antibodies were never detected by these assays.Table 1HLA status of patient and kidney and islet donorsHLA AHLA AHLA BHLA BHLA DRHLA DRMismatches1Mismatches of HLA split antigens belonging to the same broad group are not counted.Repeat mismatchesPatient230 (19)44 (12)1834n.a.2n.a.: not applicable.n.a.2n.a.: not applicable.Kidney donor #1331 (19)51 (5)8234n.a.2n.a.: not applicable.Kidney donor #2229 (19)44 (12)357–30Islet donor #13Islet yield: 106 700 IEQ.211606211450Islet donor #24Islet yield: 421 300 IEQ.232 (19)44 (12)–48101 Mismatches of HLA split antigens belonging to the same broad group are not counted.2 n.a.: not applicable.3 Islet yield: 106 700 IEQ.4 Islet yield: 421 300 IEQ. Open table in a new tab We investigated the cellular immune response of the patient at 11 years posttransplantation. We first assayed the presence of circulating regulatory T cells (T regs) in the peripheral blood by flow cytometry. The fraction of CD4+CD25high was identical to those of 5 healthy controls, but interestingly, the fraction of CD4+CD25+ T-cells expressing Foxp3+, a marker of T regs, was increased in the patient (Figure 2A). Then we assessed in vitro the T cell response against islet and second kidney donor-specific MHC antigens. The presence of cytotoxic T lymphocyte precursors (CTLp) was tested against the class I HLA antigen MM present on the islets and the kidney in a limiting dilution assay (LDA). Reactivity against class II antigens, which are not expressed by islet cells, cannot be tested by CTLp. Combinations of stimulator and target cells were used to quantify the absolute number of allospecific cytotoxic T lymphocytes (CTL) (11Kaminski E Sharrock C Hows J et al.Frequency analysis of cytotoxic T lymphocyte precursors-possible relevance to HLA-matched unrelated donor bone marrow transplantation..Bone Marrow Transplant. 1988; 3: 149-155PubMed Google Scholar,12van Kampen CA van de Linde P Duinkerken G et al.Alloreactivity against repeated HLA mismatches of sequential islet grafts transplanted in non-uremic type 1 diabetic patients..Transplantation. 2005; 81: 118-126Crossref Scopus (26) Google Scholar). CTL precursors against the HLA-A11 and B60 borne by the first islet donor and the B35 borne by the second kidney donor were detected (Figure 2B). Interestingly, the response against third party (52 6× 106) was much stronger (4- to 5-fold increase) than against kidney donor #2 and islet donor #1. A similar observation was made in a classical mixed lymphocyte reaction (MLR) experiment in which kidney #2 banked donor spleen cells were used as stimulators and patient peripheral blood mononuclear cells (PBMC) as responders (Figure 2C). No banked cells from the islet donors were available to perform the MLR. This is an exceptional case of long-term insulin independence after allogeneic islet transplantation for type 1 diabetes mellitus, and, indeed, the first to ever pass the 10-year mark, approaching 12 years posttransplantation. In addition to prolonged success in itself, it should be noted that it was achieved with a single islet infusion, a marginal mass (<10000 IEQ/kg) of transplanted islets and with persistence of normal metabolic control. Single-donor success has so far been anecdotal, and consistent insulin independence after single-donor islet transplantation has only been achieved thanks to radical peritransplant strategies aiming at improving islet mass, viability, implantation and function (13Hering BJ Kandaswamy R Ansite JD et al.Single-donor, marginaldose islet transplantation in patients with type 1 diabetes..JAMA. 2005; 293: 830-835Crossref PubMed Scopus (492) Google Scholar). Normality of metabolic control, including OGTT, is also uncommon after islet transplantation, even in insulin-independent patients (5Ryan EA Lakey JRT Rajotte RV et al.Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol..Diabetes. 2001; 50: 710-719Crossref PubMed Scopus (787) Google Scholar,6Rickels MR Schutta MH Markmann JF et al.Beta-cell function following human islet transplantation for type 1 diabetes..Diabetes. 2005; 54: 100-106Crossref PubMed Scopus (97) Google Scholar). The remarkable stability of kidney function over the years is a major achievement for this patient, especially in view of the rather poor GFR at the time of islet transplantation and is in all probability a consequence of an ideal glycaemic control (14Fiorina P Folli F Zerbini G et al.Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplantation..J Am Soc Nephrol. 2003; 14: 2150-2158Crossref PubMed Scopus (153) Google Scholar). Other micro-and macroangiopathic complications of diabetes were already severe at the time of transplant and have tended to stabilize since then. It is obviously difficult to conclude from this single case what have been the key determinants for long-term success. Our subject may have had residual beta-cell secretion, as suggested by C-peptide positivity, albeit close to the threshold of sensitivity of the assay and unresponsive to glucagon stimulation, which makes this result difficult to interpret. Residual C-peptide can be encountered in a sizeable proportion of patients with type 1 diabetes, but is associated with better long-term prognosis in terms of occurrence of nephropathy (15Steffes MW Sibley S Jackson M Thomas W et al.Beta-cell function and the development of diabetes-realted complications in the diabetes control and complications trial..Diabetes Care. 2003; 26: 832-836Crossref PubMed Scopus (561) Google Scholar), which was clearly not the case of our subject. The diagnosis of bona fide type 1 diabetes cannot be questioned in this patient who exhibited anti-GAD65 autoantibodies. Low pre-transplant insulin requirements, indicating a low metabolic demand, may also have been a favourable factor for islet engraftment. The immunosuppressive regimen administered in the ‘pre-Edmonton era’ may also have played a role. Induction with antithymocyte globulins has been recently proposed to facilitate engraftment through selective depletion of alloreactive and autoreactive T cells (13Hering BJ Kandaswamy R Ansite JD et al.Single-donor, marginaldose islet transplantation in patients with type 1 diabetes..JAMA. 2005; 293: 830-835Crossref PubMed Scopus (492) Google Scholar). Tacrolimus-free immunosuppression may have been beneficial in terms of drug diabetogenicity (16Berney T Buhler LH Majno P Mentha G Morel P et al.Immunosuppression for pancreatic islet transplantation..Transplant Proc. 2004; 36: 316-318Crossref PubMed Scopus (20) Google Scholar), and the absence of sirolimus may have been critical with respect to the alleged role of its antiproliferative properties on beta-cell regeneration (17Zahr E Molano RD Pileggi A et al.Rapamycin impairs in vivo proliferation of islet beta-cells..Transplantation. 2007; 84: 1576-1583Crossref PubMed Scopus (90) Google Scholar). Finally, low-dose steroids did not seem to be detrimental in this patient and may even have had a beneficial antiinflammatory effect. There is a theoretical possibility of islet regeneration in the native pancreas. This hypothesis could be explored by measuring C-peptide levels in the portal versus hepatic venous blood to differentiate native pancreas versus intrahepatic islet transplant as the source of insulin. However, this would be a rather invasive study that we renounced to perform. The results of the cellular in vitro assays suggest that the peripheral immune system of the recipient is able to mount a CTL response against antigenic determinants borne by the islets isolated from the first donor and the second kidney transplant. However, cytotoxic and proliferative cellular responses weaker than against third party suggest a state of hyporesponsiveness against donor antigens. Higher Foxp3 expression in CD4+CD25+ T cells as compared to controls suggests that regulatory T cells may be involved in the islet graft long-term survival. This observation is so far an isolated and exceptional case, but clearly demonstrates the validity of the concept that long-term insulin independence after allogeneic islet transplantation is an achievable target in the therapy of type 1 diabetes mellitus. We will certainly soon face several such cases in the near future, when the 10–15% patients transplanted since the turn of the century and who are still off-insulin reach the symbolic 10-year mark. The availability of such a cohort of patients will call for the in-depth examination and understanding of the singular features of such individuals, in order to be able to reproduce these results in a larger scale and turn into the standard what is still as yet the exception. This study was partly funded thanks to the continuous support of the Swiss National Foundation for Scientific Research (Grant No to and The had no role in the and of the the analysis and of the or the or of the of after allogeneic islet transplantation for type 1 over the 10-year by et Am J Open

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