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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
Chapter 1
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.
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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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Introduction
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].
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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. Betacells 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.
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Risks
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].
Injection risks,
teratoma risk
Injection risks,
teratoma risk
Injection risks,
zoonosis
Operation risks,
transmittable
infections
Injection risks,
transmittable
infections
Injection risks,
tumor risk
Injection risks,
transmittable
infections, genetic
instability?
Donor
dependent
Controllable
until pancreatic
endoderm stage
Inter animal
variation
dependent
Donor and
procedure
dependent
Quality
consistency
Donor and
procedure
dependent
Controllable
Invasive injection
Invasive
injection
Invasive
injection
Invasive
injection
Major operation
Transplant
Procedure
Invasive
injection
Invasive
injection
None
None
Phase I/II trial
ongoing
Early studies
Extensive
Clinical
Experience
Extensive
None
Moderate or limited
Unlimited
Unlimited
Unlimited
Limited
Availability
Very limited
Unlimited
Transdifferentiation
from other cell
types*
iPS cells*
Pigs
Pancreas
Islets
Beta-cell line
hESC
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Introduction
Donor and procedure
dependent
Chapter 1
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].
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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.
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Chapter 1
xenotransplantation) or through direct toxic effect of some inflammatory cytokines
on beta-cells [11,25,58,74].
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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 betacells 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.
Introduction
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].
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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 riskbenefit 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 betacell immunity is examined.
Immune suppression for beta-cell transplantation generally consists of aggressive
immune suppression at moment of transplantation (induction therapy), followed by
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