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The handle http://hdl.handle.net/1887/47907 holds various files of this Leiden University dissertation.
Author: Torren, C.R. van der
Title: Investigating remission and relapse in type 1 diabetes. Immune correlates of clinical outcome in beta-cell replacement therapies
Issue Date: 2017-04-12
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General Introduction
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Chapter 1 Introduction
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TREATMENT OF TYPE 1 DIABETES
Several drugs can increase tissue sensitivity to insulin and therefore treat type 2 diabetes, but type 1 diabetes can only be treated with additional insulin. Insulin can be injected several times a day in a combination of directly acting and delayed response insulin analogs. This is usually done before meals to allow compensation for the intake at meals and sufficient insulin for activities between meals. More recently, continuous insulin pumps have become popular, which continuously inject basal insulin and can be adjusted for intake and activity. The next step will be an
‘artificial pancreas’, which adjusts automatic insulin infusion by continuous glucose measurement. Recent results are promising although technically challenges remain [153,283]. With current therapy, however, only 20% of patients with type 1 diabetes are reported to achieve international goals for glucose control [27].
Ideally, type 1 diabetes would be treated with beta-cells, since lack of beta-cells is the cause of the disease and they combine glucose regulated insulin secretion with their own insulin production. At time of type 1 diabetes diagnosis there may be sufficient beta-cells left. Patients can become independent of insulin injections for a short time after onset of therapy, called the honeymoon phase [292]. However, protecting remaining beta-cells from further autoimmune destruction has shown to be very challenging. So far, cure has only been achieved by maximally aggressive immune suppressive therapy which requires consecutive hematopoietic stem cell transplantation to survive [307]. When insufficient beta-cells remain, beta-cells may be transplanted.
BETA-CELL TRANSPLANTATION
Beta-cell transplantation is a technical as well as immunological challenge. Beta- cells reside in the islets of Langerhans, in short islets, which are clusters of endocrine (hormone producing) cells located throughout the pancreas. These islets compose 1-2% of the pancreas, which further produces digestive (exocrine) proteins. The islets are composed of endocrine alpha-, beta-, delta-, PP- and epsilon- cells. The majority are insulin producing beta-cells. Alpha-cells produce glucagon, which counters insulin by stimulating release of glucose from tissue and gluconeogenesis. The other cells produce various regulatory hormones that influence energy homeostasis including affecting insulin and glucagon release. Currently, beta-cells from organ donors are transplanted together with the whole pancreas or in isolated islets.
Pancreas transplantation is usually preferred for long term outcome with 80%
pancreas graft survival after 3 years in major cohorts [102]. Pancreas transplantation can be performed separately, but often it is combined with kidney transplantation or follows after kidney transplantation for diabetic kidney failure. The combination with kidney transplantation usually improves outcome of both pancreas and kidney graft, possibly because of better graft condition or because rejection monitoring for the kidney is easier [102,152]. Pancreas transplantation also has major disadvantages, since it requires major abdominal surgery with consequential morbidity and mortality.
Also, the exocrine part of the pancreas needs to drain to the bowel or bladder and can cause anastomosis leakage, pancreatitis and irritation of the bladder wall.
I am writing these first words of my thesis only a few days after I have made my first diagnosis of type 1 diabetes. A child, only three years old, with impressive deep (Kussmaul) breathing as part of diabetic ketoacidosis. For this severe complication of her diabetes she was referred to the intensive care department. Her mother’s joy of her quick recovery was heartening; although this was only the beginning of her life with a challenging disease.
DIABETES
Diabetes develops from insulin insufficiency, which leads to inadequate uptake and processing of glucose and fat in cells. This results in high blood glucose levels (hyperglycemia). In the Netherlands, five percent of the population has diabetes and the incidence is steadily rising (rivm.nl). Most patients have decreased sensitivity of cells to insulin and therefore a relative insulin shortage; type 2 diabetes. Some patients have diabetes temporarily during pregnancy, while a minority of patients has rare genetic, disease or drug induced diabetes. This thesis focusses on patients with an autoimmune attack on insulin producing beta-cells causing absolute insulin shortage; type 1 diabetes. This constitutes 10% of all patients with diabetes and is the most frequent cause of diabetes in children.
Diabetes can lead to several complications of which most arise gradually in years up to decades and can be prevented by excellent regulation of blood glucose levels [2,4]. These complications are cardiovascular disease, which may lead to heart attack and stroke; kidney damage (nephropathy), which may ultimately require kidney transplantation; eye damage (retinopathy), which can lead to blindness; and nerve damage (neuropathy), with loss of feeling in the feet causing difficulty walking and risk of severe wounds. When insulin shortage is extreme (e.g. unrecognized or untreated type 1 diabetes), cells take up insufficient glucose to survive and cells start degrading alternative energy sources. This alternative burning produces acidic ketones and ultimately leads to ketoacidosis (acidic blood due to ketones), which can be life threatening. In addition, treatment related complications can arise including side effects of drugs and hyper- or atrophy of subcutaneous fat at site of insulin injections. Most importantly, too much insulin can reduce blood glucose to dangerously low levels (hypoglycemia), which untreated may ultimately result in convulsions, coma and even death.
Tight regulation of blood glucose levels is therefore essential, but has a major impact on daily life of patients with diabetes. For them, glucose regulation requires insulin injections. All caloric intake (meals, snacks, even drinks) needs to be calculated and affects the required amount of insulin. Adjustments are also needed for daily activities, like sports, which influence the need for energy and insulin sensitivity. Additionally, blood glucose measurements are required several times per day (including at night) to optimize insulin injections for tight glucose control and prevention of hypoglycemia.
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Islets transplantation has the major advantage that an abdominal operation is not required since only the endocrine islets of the pancreas are transplanted. These islets are generally infused into the portal vein and then spread throughout the liver.
To procure the islets, a donor pancreas needs to be digested after which the islets are purified. Next, islets can be maintained in culture for planned transplantation and further purification before injection. However, islet yields are often suboptimal while digestion and purification of the pancreas can affect beta-cell quality. This results in transplantation of islets from only 1 in 2 organs, while multiple organs may be required to achieve insulin independence [135,255]. Additionally, in the Eurotransplant region only pancreases declined for whole organ transplantation are available for islets isolation, since the procedure is considered less successful and cost-effective [89].
Nonetheless, insulin independence can be achieved in most patients and long term outcome has been steadily improving over the years [18,150].
The primary restriction for pancreas and islet transplantation is the limited availability of donor organs. Alternative sources of beta-cells are therefore required to make beta-cell transplantation a success. Attempted approaches to acquire beta-cells are regeneration by proliferation of beta-cells or differentiation from other cell types; differentiation from (induced) stem cells; and isolation of animal islets. These approaches have varying success in the experimental setting, while source specific availability and safety issues have to be taken into account (Table 1.1). Beta-cell lines with induced proliferation provide pure beta-cells, but transduction with oncogenes creates a tumor risk that precludes clinical application [224]. Embryonic stem cells currently need in vivo differentiation to acquire beta-cells mixed with other pancreatic endocrine cells. Currently, efficacy of this differentiation process in humans is investigated in a phase 1/2 trial (clinicaltrials.gov, NCT02239354). Also, the potential risk of teratoma growth from stem cells requires great precaution, which may require containment in capsules for transplantation (viacyte.com) [250]. Beta-cells isolated from pig pancreases may be equally effective in humans, but may contain viruses which could potentially adept to humans after transplantation [318]. Apart from these health risks, the origin of these beta-cells involves ethical issues which requires consideration of acceptance by society and potential recipients [157,226,268,308].
IMMUNE RESPONSES IN BETA-CELL TRANSPLANTATION
Transplanted beta-cells are at risk of direct and indirect immune attack, which may result in their destruction. Inflammation is induced by surgical damage or graft damage through hypoxia or islet isolation procedure, while direct exposure of islets to blood can induce instant blood mediated inflammatory reaction (IBMIR) [25]. Damaged cells are marked by immune complement factors, which attract inflammatory cells. These inflammatory cells produce signal molecules (cytokines and chemokines), thereby recruiting more inflammatory cells and regulating the immune response. Complement marked cells can be taken up by antigen presenting cells, which present them to T-cells and can start an adaptive immune response.
Beta-cells may also endure direct damage in this phase from excessive coagulation in IBMIR, by natural killer (NK-)cell attack if not recognized as human cell (e.g.
PancreasIsletsPigsBeta-cell linehESCiPS cells*Transdifferentiation from other cell types* AvailabilityLimitedVery limitedUnlimitedUnlimitedUnlimitedUnlimitedModerate or limited
Clinical Experience ExtensiveExtensiveEarly studiesNonePhase I/II trial ongoingNoneNone
Transplant Procedure
Major operation
Invasive injection Invasive injection Invasive injection Invasive injection Invasive injection
Invasive injection
Quality consistency Donor and procedure dependent Donor and procedure dependent
Inter animal variation dependent
ControllableControllable
until pancreatic endoderm stage
Donor dependentDonor and procedure dependent Risks
Operation risks, transmittable infections
Injection risks, transmittable Injection risks, Injection risks, zoonosistumor risk infections Injection risks, teratoma risk Injection risks, teratoma risk Injection risks, transmittable
infections, genetic instability? Table 1.1. Overview of alternative beta-cell sources. hESC: human embryonic stem cells; iPS cells: induced pluripotent stem cells. *No experimental data on human beta-cells available [18,102,150,152,157,226,248,250,255,268,308,318].
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Chapter 1 Introduction
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maintenance immune suppression for the lifetime of the graft. Side effects of immune suppression include increased infection risk and risk of cancer. Side effects are a major limitation for transplantation to younger patients and patients with fewer diabetic complications. Optimization of immune suppression in terms of efficacy versus side effects is necessary to allow broader application of beta-cell transplantation for diabetes. Other drug combinations may allow better protection from recurrent autoimmunity, since current protocols have been adjusted from kidney transplant protocols which only need to prevent alloreactivity. Another major improvement would be induction of graft tolerance, which would allow reduction (tapering) and possibly discontinuation of immune suppression. Such cases have been described for liver and kidney transplantation, although not yet for beta-cell transplantation [77,109,200]. In future, immune protective encapsulation may abrogate the need for general immune suppression. This strategy has been under investigation for a long time, but the desire to contain alternative beta-cells on transplantation has given this field new ambition [28,147].
AIMS OF THIS THESIS
Type 1 diabetes can only be cured through beta-cells, which then require adequate immune protection. For most patients with type 1 diabetes, current intervention and transplantation therapies have insufficient efficacy or too many side effects to challenge insulin therapy. Immune suppression heavily contributes to both efficacy and side effects. This thesis aims to uncover opportunities to improve the risk- benefit ratio for beta-cell immune protection. Chapter 2 addresses the challenge to preserve a patient’s remaining beta-cells. Chapter 3 explores immune infiltration of transplanted islets in their liver environment. Chapter 4 describes discovery of novel immune biomarkers to help improve beta-cell transplantation. Opportunities to reduce side effects of immune suppression are investigated in Chapter 5. Finally, in Chapter 6, the potential of novel beta-cell sources to explore and overcome beta- cell immunity is examined.
xenotransplantation) or through direct toxic effect of some inflammatory cytokines on beta-cells [11,25,58,74].
Antigen presenting cells and T-helper cells control the progression of the immune response. Antigens are presented to T-cells in the human leukocyte antigen (HLA) molecule. All cells present internal antigens through HLA (class I) to cytotoxic T-cells for immune surveillance against intracellular infections. Antigen presenting cells additionally present antigens taken up from external material in HLA (class II) to T-helper cells. If T-helper cells recognize their antigen on an antigen presenting cell they can be activated to either pro-inflammatory (Th1- or Th2-) or regulatory (Treg) T-cell. Th-cells will help to activate B-cells and cytotoxic (killer) T-cells, while Tregs will prevent immune responses to the antigen (usually self-derived antigens) and therefore prevent autoimmunity or impose tolerance. At onset of type 1 diabetes Tregs must have failed to contain an immune response against beta-cell antigens, leading to autoimmune destruction of these beta-cells.
The adaptive immune response kills through marking cells by antibodies for attack by innate immune cells and complement or direct recognition and killing by cytotoxic T-cells. For transplanted cells, differences between donor and recipient are recognizable patterns for an immune response (alloreactive response). HLA itself is a major target, since it is highly variable between individuals and exposed to the recipient’s immune system. Alloreactive cytotoxic T-cells can respond to the different HLA molecule directly or to donor antigen in an HLA molecule that match the recipient’s HLA [99,299]. Similarly, the HLA differences are a target for alloreactive antibodies. For most organ transplantations, HLA of donor and recipient is matched to prevent allograft rejection. However, HLA matching facilitates recognition of beta- cells by memory T-cells in beta-cell transplantation to patients with type 1 diabetes.
Memory T-cells and B-cells effectively prevent recurrent infection, but may also lead to recurrence of autoimmunity. Presence of autoimmune T-cell before islet transplantation often results in unsuccessful islet transplantation [124]. Recurrent autoimmunity has also been recognized after pancreas transplantation [35,259,276,291]. The significance of autoimmune antibodies in beta-cell transplantation is unclear, but may depend on immune suppression [124,212]. Known beta-cell specific antibodies are directed at intracellular molecules, suggesting their effect may be indirect through enhanced presentation of antigens from dead beta-cells [312]. Alternatively, they may be a byproduct of the autoimmune response, which has been suggested for autoimmune antibodies at onset of type 1 diabetes [168]. In contrast, presence of alloreactive antibodies, from pregnancy, blood transfusion or a previous transplant, can cause (hyper)acute rejection of the transplanted organ [51,281]. This does not only additionally complicate transplantation for these patients, but may also pose a risk for patients who are unsuccessfully transplanted and may need another transplant in the future. The challenge of immune memory for transplantation is further emphasized by recognizing that immune responses mentioned above infringe the graft despite immune suppression.
Immune suppression for beta-cell transplantation generally consists of aggressive immune suppression at moment of transplantation (induction therapy), followed by
