Allogeneic islet transplantation is a standard of care treatment for patients with labile type 1 diabetes in many countries around the world, including Japan, the United Kingdom, Australia, much of... Show moreAllogeneic islet transplantation is a standard of care treatment for patients with labile type 1 diabetes in many countries around the world, including Japan, the United Kingdom, Australia, much of continental Europe, and parts of Canada. The United States is now endorsing islet cell treatment for type 1 diabetes, but the FDA has chosen to consider islets as a biologic that requires licensure, making the universal implementation of the procedure in the clinic very challenging and opening the manufacture of islet grafts to private companies. The commercialization of human tissues raises significant legal and ethical issues and ironically leads to a situation where treatments developed as a result of the scientific and economic efforts of academia over several decades become exploited exclusively by for-profit entities. Show less
Nano, R.; Kerr-Conte, J.A.; Scholz, H.; Engelse, M.; Karlsson, M.; Saudek, F.; ... ; Piemonti, L. 2020
Background. Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further... Show moreBackground. Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries.Methods. A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan.Results. Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities.Conclusions. The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria. Show less
Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of... Show moreTransplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity. Show less
BACKGROUND The safety of any immune modulating agent in type 1 diabetes mellitus (T1DM) involves its selectivity on autoimmunity and its preservation of recall and tumour immunity. METHODS We... Show moreBACKGROUND The safety of any immune modulating agent in type 1 diabetes mellitus (T1DM) involves its selectivity on autoimmunity and its preservation of recall and tumour immunity. METHODS We performed lymphocyte proliferation tests on seven recent onset diabetic patients treated with anti-CD3 (Otelixizumab; ChAglyCD3) and five recent onset diabetic patients treated with placebo, on average 2 years after therapy. RESULTS Proliferative responses towards common viral, bacterial and yeast antigens upon in vitro stimulation with a range of recall antigens in anti-CD3-treated T1DM patients were highly similar to those in placebo-treated T1DM patients. Similarly, T-cell responses towards autoantigens were equally low between the two groups, several years after diagnosis of T1DM. The proliferative response upon stimulation with the human suppressor protein p53 was invariably high in both anti-CD3- and placebo-treated patients, implying preserved anti-tumour immunity in anti-CD3 treatment. CONCLUSIONS As long-term focus on side effects is key, we demonstrate in this sub-cohort of recent onset T1DM patients treated with Otelixizumab that recall immunity is preserved in spite of high-dose anti-CD3 treatment, adding to the safety of anti-CD3 treatment as an immune-modulatory agent in the treatment of T1DM. Show less
Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is... Show moreAutoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes. Show less
OBJECTIVE Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a... Show moreOBJECTIVE Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8(+) T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B(10-18), prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(265-273), and prepro islet amyloid polypeptide (ppIAPP)(5-13)-specific CD8(+) T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B(10-18), IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI(15-24) proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples. Show less
OBJECTIVE-Type I diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a... Show moreOBJECTIVE-Type I diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8(+) T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS-Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10-18, prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123) islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(265-973), and prepro islet amyloid polypeptide (ppIAPP)(5-13)-specific CD8(+) T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS-Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B10-18, IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15-24 proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS-A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples. Diabetes 59:1721-1730, 2010 Show less
