Title: Investigating remission and relapse in type 1 diabetes. Immune correlates of clinical outcome in beta-cell replacement therapies

Cover Page

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

C.R. van der Torren

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Abatacept Teplizumab

Otelixizumab IL1 Anakinra

Diamyd DiaPep IL1R Rituximab

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Autoantibodies ₀

25 50 75 100

PRA

50 100 150 200

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Otelixizumab III Azathioprine

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Complication resistant patients Complication

prone patients

Investiga ting Remission and Relapse in Type 1 Diabetes C.R. v an der Torren

Investigating Remission and Relapse in Type 1 Diabetes

Immune Correlates of Clinical Outcome

in Beta-Cell Replacement Therapies

Stellingen behorende bij het proefschrift

INVESTIGATING REMISSION AND RELAPSE IN T1D

Beta-cell Replacement Therapies

1. Beta-cell immune protection is the foundation of any cure for type 1 diabe- tes.

(this thesis)

2. Monitoring of auto- and alloreactive immune responses can act as safety measure for encapsulated alternative beta-cell transplantation.

(this thesis)

3. Immune biomarkers predicting favorable clinical outcome in beta-cell trans- plantation are currently ignored.

(this thesis)

4. Clinical islet transplantation has evolved beyond the experimental stage, be it at a price.

(this thesis)

5. One size does not fit all.

(this thesis)

6. Cure of type 1 diabetes at onset is possible.

7. Children have the greatest unmet medical needs of patients with type 1 diabetes.

8. Cadaveric pancreases and islets remain insufficient to meet the demands in beta-cell transplantation.

9. Immune suppression prevents graft rejection, but impairs immune toler- ance.

10. Being sure in science is being dogmatic.

11. Yesterday is not ours to recover, but tomorrow is ours to win or lose.

(Lyndon B. Johnson, 1963) 12. It’s all a matter of perspective.

Investigating Remission and Relapse in Type 1 Diabetes

Immune Correlates of Clinical Outcome in

Beta-Cell Replacement Therapies

Investigating Remission and Relapse in Type 1 Diabetes

Immune Correlates of Clinical Outcome in Beta-Cell Replacement Therapies

Proefschrift

ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. C.J.J.M. Stolker,

volgens besluit van het College voor Promoties te verdedigen op woensdag 12 april 2017

klokke 13:45 uur

door

Cornelis Rudolf van der Torren geboren te Heerlen in 1984 Cover design and lay-out: C.R. van der Torren

Cover image: designer MedicalGraphics.de (license: CC BY-ND 3.0 DE) Printed by: Gildeprint

ISBN: 978-94-6233-584-4

©2017 C. R. van der Torren.

All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of copyright owner.

Contents

Chapter 1. General Introduction

Chapter 2. Immune intervention trials towards a cure for type 1 diabetes De (om)Weg naar Genezing van Type 1 Diabetes 17 Chapter 3. Fate of Transplanted Islets, Local Insights

Identification of Donor Origin and Condition of Transplanted

Islets in situ in the Liver of a Type 1 Diabetic Recipient 29 Chapter 4. Immune Biomarkers of Islet Transplantation Outcome

A Pre-Transplant GAD-Autoantibody Status to Guide Prophylactic Antibody Induction Therapy in Simultaneous

Pancreas and Kidney Transplantation. 45

B Serum Cytokines as Biomarkers in Islet Cell Transplantation

for Type 1 Diabetes 59

Chapter 5. Consequences of Reducing Immunosuppressive Burden in Islet Transplantation

A Immune Responses against Islet Allografts during Tapering of

Immunosuppression - a Pilot Study in 5 Subjects 75 B Predictive Factors of Allosensitization following

Immunosuppressant withdrawal in Recipients of Long-Term

Cultured Islet Cell Grafts 89

Chapter 6. Immunogenicity of Beta-Cells from Alternative Sources A Innate and Adaptive Immunity to Human Beta-Cell Lines:

Implications for Beta-Cell Therapy 105

B Immunogenicity of Human Embryonic Stem Cell-derived

Beta-Cells 115

Chapter 7. General Discussion

R References & Abbreviations 141

& Summary 161

& Samenvatting 165

& Acknowledgements 169

& Curriculum Vitae 170

& Publications 171

PROMOTOREN

Prof.dr. P. Gillard (Katholieke Universiteit Leuven, Belgium)

PROMOTIECOMMISSIE

Dr. A.A. van Apeldoorn (Maastricht University)

Prof.dr. K. Buschard (The Bartholin Institute, Copenhagen, Denmark)

The work presented in this thesis was supported by the European Commission (FP6 and BetaCellTherapy, number 241883 in the FP7 program), Dutch Diabetes Research Foundation, the Juvenile Diabetes Research Foundation.

1

General Introduction

10 11

1

Chapter 1 Introduction

1

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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Chapter 1 Introduction

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].

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

1

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

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Immune intervention trials towards a cure for

type 1 diabetes

Nederlands Tijdschrift voor Geneeskunde 2012;156(15):A4268

Cornelis R. van der Torren en Bart O. Roep

De (om)Weg naar Genezing van Type 1 Diabetes

Chapter 2

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Chapter 2 Immune Intervention Trials towards a Cure for Type 1 Diabetes

2

Bij slechts 20% van de patiënten met diabetes mellitus type 1 lukt het om de gewenste bloedsuikerwaarden te handhaven. Om dit te verbeteren doet men tegenwoordig veel moeite om de eigen bètacelfunctie van de patiënt te behouden of te herstellen;

hierbij moet de auto-immuundestructie van de insuline-producerende bètacellen worden gestopt.

Na bemoedigende resultaten van verschillende klinische fase 2-trials met immunotherapie voor patiënten bij wie kort daarvoor diabetes mellitus type 1 was gediagnosticeerd, bleken afgelopen jaar de resultaten van de eerste fase 3-trials tegen te vallen. Dit was enerzijds het gevolg van te hoog gespannen verwachtingen, maar anderzijds hadden de klinische trials een inadequate studieopzet. De behandeling van diabetes mellitus type 1 zal zich desondanks de komende tijd sterk blijven ontwikkelen. In de toekomst zal de behandeling van diabetes mellitus type 1 niet alleen gericht zijn op de symptomen van bètaceldestructie, met insulinesuppletie, maar waarschijnlijk ook de oorzaak van deze ziekte aanpakken in de vorm van immunotherapie.

HET PROBLEEM

De ontdekking van insuline, nu 90 jaar geleden, heeft diabetes mellitus type 1 veranderd van een dodelijke ziekte in een chronische aandoening met aanzienlijke morbiditeit en mortaliteit. Ontwikkelingen in de productie van insuline, insuline- analogen en hulpmiddelen voor insulinetoediening hebben, samen met verbeterde technieken voor glucosemonitoring, de kwaliteit van leven voor patiënten met diabetes mellitus type 1 sterk verbeterd. Naar aanleiding van de resultaten van de ‘Diabetes control and complication’ (DCCT)-studie en de ‘United Kingdom prospective diabetes study’ is de insulinebehandeling geïntensiveerd, waardoor het ontstaan van langetermijncomplicaties bij veel patiënten kan worden vertraagd of mogelijk voorkómen [2,4].

Ondanks de verbeterde therapie legt diabetes mellitus type 1 een continu beslag op het leven van patiënten. Daarbij komt de zorg voor eventuele complicaties. De strikte glucosecontrole die nodig is om complicaties te voorkomen – met de streefwaarde HbA1c <7% (53 mmol/mol) volgens de huidige richtlijnen – blijkt voor minder dan 1 op de 5 volwassen patiënten haalbaar. Bij kinderen en adolescenten is strakke regulatie nog moeilijker [27]. Dat een goede glucosecontrole langdurig consequenties heeft, blijkt uit een groeiend verschil in complicaties tussen de interventiegroepen in de DCCT-trial, lang na afloop van de trial [191]. Behoud van enige endogene insulineproductie bevordert de glucoseregulatie met exogene insulinetherapie en helpt waarschijnlijk complicaties op de lange termijn te voorkomen, zoals blijkt uit gedeeltelijke regressie van complicaties bij patiënten die een pancreas- of eilandjestransplantatie hebben ondergaan [100,101,132,286].

ABSTRACT

● Treatment of type 1 diabetes mellitus has greatly improved but remains limited ● to combating the consequences of the disease;

● Target values for glucose regulation are achieved in only 20% of patients;●

● Immunosuppression can slow disease progression, but does not cure type ● 1 diabetes mellitus. Immunotherapy attempts to protect remaining insulin- producing beta-cells and beta-cell function;

● Promising results of immunotherapy in phase 2 studies in patients with type 1 ● diabetes mellitus could not be reproduced in phase 3 studies. These studies showed heterogeneity played a role in patient populations and between ethnic groups;

● In future studies better endpoints of efficacy, biomarkers of disease progression ● and response to therapy are essential;

● Vaccination with beta-cell specific antigens to stimulate tolerance and vaccination ● combined with immunotherapy (biologicals) are options for future therapy;

● Discussion on the acceptability of the side effects of immunotherapy is desirable.●

SAMENVATTING

● De behandeling van diabetes mellitus type 1 is sterk verbeterd, maar beperkt ● zich tot bestrijding van de gevolgen van de ziekte;

● De streefwaarden voor glucoseregulatie worden bij slechts 20% van de patiënten ● behaald;

● Immuunsuppressie vertraagt het ziekteproces, maar geneest diabetes mellitus ● type 1 niet. Immunotherapie streeft naar behoud van insulineproducerende bètacellen en bètacelfunctie;

● Veelbelovende resultaten met immunotherapie in fase 2-studies bij patiënten ● met diabetes mellitus type 1 konden niet worden gereproduceerd in fase 3-studies. Uit deze studies bleek dat heterogeniteit in patiëntenpopulaties en tussen etnische groepen een rol speelt;

● Voor toekomstige studies is behoefte aan betere uitkomstmaten voor effectiviteit ● en aan biomarkers voor ziekteprogressie en voor de respons op de therapie;

● Vaccinatie met bètacelspecifieke antigenen om tolerantie op te wekken en ● combinatie hiervan met immunotherapie (biologicals) zijn opties voor een toekomstige therapie;

● Discussie over de aanvaardbaarheid van bijwerkingen van immunotherapie bij ● diabetes mellitus type 1 is gewenst.

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Chapter 2 Immune Intervention Trials towards a Cure for Type 1 Diabetes

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Vooralsnog werd aangenomen dat de ziekte zich manifesteert als 80% van de bètacelmassa is vernietigd. Deze celmassa wordt geschat op basis van de endogene bètacelfunctie, die wordt bepaald aan de hand van de serumconcentratie C-peptide (een afbraakproduct van endogeen geproduceerd insuline) na het stimuleren van de insulineafgifte met bijvoorbeeld een gestandaardiseerde maaltijd [111]. Uit recente autopsiestudies bij patiënten met diabetes mellitus type 1 blijkt echter dat de ziekte bij mensen zeer heterogeen is [59].

Na het stellen van de diagnose ‘diabetes mellitus type 1’ zijn bij sommige patiënten nog aanzienlijke aantallen bètacellen aanwezig en zelfs decennia later kunnen nog normaal ogende eilandjes van Langerhans in de pancreas worden aangetroffen [59,314]. De bètaceldisfunctie is omkeerbaar, wat blijkt uit een tijdelijke opleving bij veel patiënten van de endogene insulineproductie in de ‘honeymoon’-fase, direct na het begin van de insulinetherapie. Deze opleving is helaas van korte duur, aangezien het auto-immuunproces nog actief is. In deze fase kan agressieve immunotherapie zelfs tot volledige en langdurige remissie leiden [60].

IMMUNOTHERAPIE VOOR DIABETES MELLITUS TYPE 1

Hoewel diverse immunotherapieën voor diabetes mellitus type 1 effectief bleken, is de ontwikkeling van een standaardtherapie niet eenvoudig. Reeds in de jaren

‘80 van de vorige eeuw werd aangetoond dat immuunsuppressie van T-cellen met bijvoorbeeld cyclosporine of azathioprine het beloop van diabetes mellitus type 1 kon vertragen, maar de bijwerkingen verhinderden langdurig gebruik. Bovendien bleek dat immuunsuppressieve therapie bij transplantatie van eilandjes van Langerhans naar patiënten met diabetes mellitus type 1 onvoldoende bescherming bood tegen bestaande auto-immuniteit tegen eilandjes [124].

Toch is het mogelijk om patiënten te genezen van diabetes mellitus type 1 met een controversiële, rigoureuze immunotherapie met autologe hematopoëtische stamcellen na agressieve inductietherapie met cyclofosfamide en thymocytenimmunoglobuline.

In een onderzoek bij 23 patiënten waren er nog 12 insuline-onafhankelijk na gemiddeld 30 maanden follow-up [60]. Hoewel deze studie een ‘proof of concept’

levert dat genezing met behulp van immuuninterventie therapie mogelijk is zonder bètacelvervanging, lijken ook hier de bijwerkingen en risico’s op lange termijn niet op te wegen tegen de eventuele complicaties als gevolg van ontoereikende insulinetherapie.

BIOLOGICALS

De zoektocht naar een kortdurende en gerichtere immuuninterventie met langdurig effect gaat verder, en is in de afgelopen jaren in een stroomversnelling geraakt toen bleek dat biologicals in verschillende fase 2-studies de bètacelfunctie tijdelijk beschermden. Deze behandelingen richtten zich tegen T-cellen, met de gemodificeerde anti-CD3-antilichamen teplizumab en otelixizumab [114,139], tegen B-cellen, met het anti-CD20-antilichaam rituximab [209], en tegen immuunactivatie door co-stimulatie van T-cellen te blokkeren met abatacept, een anti-CTLA-4- immuunglobuline [198]. Hoewel patiënten in deze studies doorgaans afhankelijk β

CD80/86

α B

T

APC T

β

β β CTL

CD28 CTLA4

TCR

α

HLA Antigeen

AutoAb

Eilandje van Langerhans

Tn

β

Abatacept Teplizumab

Otelixizumab

IL1 Anakinra

Diamyd DiaPep IL1R

Rituximab

β

Figure 2.1. Schematische weergave van de pathogenese van diabetes mellitus type 1 en interventies die aangrijpen op het auto-immuunproces. Antigenen afkomstig uit insulineproducerende bèta-cellen (β) worden door antigeenpresenterende cellen (APC) opgenomen en afgevoerd. Door activatie van specifieke, autoreactieve pro-inflammatoire T-cellen (Th1) kan een auto-immuunreactie worden opgewekt, waarbij B-lymfocyten (B) worden aangezet tot productie van autoantilichamen (autoAb) en cytotoxische T-lymfocyten (CTL) tot het herkennen en vernietigen van bèta-cellen die het antigeen via HLA presenteren. Bij de herkenning is de T-celreceptor (TCR) betrokken. Teplizumab en otelixizumab zijn antilichamen tegen T-cellen; rituximab is een antilichaam tegen B-cellen. De activatie van ongeactiveerde (‘naïeve’) T-cellen (Tn) kan worden voorkomen met abatacept. De activatie van Th1- cellen wordt tegengegaan door anakinra, een interleukine-1-receptorantagonist. Regulatoire T-cellen (Treg) die specifiek zijn voor het antigeen van de bèta-cellen, remmen de auto-immuuncascade die leidt tot vernietiging van de bèta-cellen. Vaccinatie met glutamaatdecarboxylase (GAD65) of een heat-shock protein 60-peptide (hsp60) induceert deze regulatoire T-cellen. Deze vorm van vaccinatie wordt wel tolerogene vaccinatie genoemd.

AANKNOPINGSPUNTEN VOOR THERAPIE

Diabetes mellitus type 1 is het gevolg van auto-immuundestructie van de insulineproducerende pancreatische bètacellen in de eilandjes van Langerhans (Figure 2.1) [241].

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Chapter 2 Immune Intervention Trials towards a Cure for Type 1 Diabetes

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het immuunsysteem zelf worden gereguleerd. Regulatoire T-cellen kunnen immuunreacties tegen specifieke auto-antigenen tegengaan zonder de reactie tegen pathogenen aan te tasten en zo specifieke tolerantie induceren. Terwijl bij gebruikelijke vaccinaties de specifieke antigenen worden geïnjecteerd in combinatie met een activerend adjuvans om een sterke immuunrespons op te wekken, wordt bij tolerogene vaccinatie het auto-antigeen toegediend zonder adjuvans of in een zogenoemde modulerende context, zoals op het oppervlak van regulatoir gemodificeerde, antigeenpresenterende cellen.

De eiwitten glutamaatdecarboxylase (GAD) en heat-shock protein 60 (hsp60) zijn geassocieerd met diabetes mellitus type 1. Tolerogene vaccinaties met de isovorm GAD65 en een peptide van hsp60 vertraagden in fase 2-studies het verlies van bètacelfunctie [159,225]. Ook hierbij gaf een fase 3-studie andere inzichten; in 2011 werd bekend dat GAD vaccinatie in de fase 3-studie de afname van C-peptide, een maat voor de bètacelfunctie, niet significant vertraagde [160]. Ook een recente fase 2-studie met vergelijkbare opzet liet geen significant effect van vaccinatie met GAD zien [313].

Het is nog onduidelijk waarom de resultaten van de initiële studie niet bevestigd konden worden; de aandacht gaat nu uit naar subgroepen waarin significante effectiviteit werd bereikt. Bovendien wordt bestudeerd welke nadelige effecten gelijktijdige griepvaccinaties (N1H1) kunnen hebben gehad op tolerantie-inductie. In de fase 2-studie werden patiënten vooral in het voorjaar geïncludeerd, terwijl in de fase 3-studie inclusies vooral in september plaatsvonden.

Het bedrijf dat het hsp60-peptide ontwikkelt, maakte eind november 2011 bekend dat hun fase 3-studie wel succesvol verliep (www.andromedabio.com); met dit middel werd bescherming van de bètacelfunctie en verbetering van de HbA1c-waarden gezien. In 2011 is een 2e fase 3-studie met dit middel van start gegaan.

TOEKOMST VOOR DE BEHANDELING

Op dit moment lopen meerdere klinische trials naar immunotherapie voor diabetes mellitus type 1. Hoewel sommige experimentele behandelingen veelbelovend zijn, blijkt het moeilijk een standaardtherapie voor alle patiënten te ontwikkelen. Recente klinische trials geven belangrijke lessen voor de toekomst [238]. Uit vrijwel alle studies komt naar voren dat ingrijpen snel na de diagnose vereist is om een effect te kunnen meten, wat overigens niet impliceert dat interventie in een later stadium niet zinvol kan zijn.

UITKOMSTMATEN

De juiste uitkomstmaten zijn essentieel voor een goede interpretatie van de resultaten.

HbA1c is een discutabele marker voor de gemiddelde bloedglucosewaarde op individueel niveau, zo bleek uit recent onderzoek met continue bloedglucosemeting [315]. De HbA1c-waarde wordt beïnvloed door allerlei factoren, waaronder leeftijd, etniciteit en comorbiditeit. Overigens mag HbA1c misschien niet als primaire uitkomstmaat van immunotherapie gelden, aangezien deze parameter het effect bleven van insuline, is de verwachting dat dit behoud van eigen bètacelfunctie

tot betere regulatie van bloedsuikerconcentraties op lange termijn leidt. Of de bescherming aanhoudt, moet nog blijken, maar bij patiënten behandeld met anti- CD3-antilichamen waren de insulinebehoefte en de HbA1c-waarden na 4 jaar lager dan in de controlegroep [139].

Dergelijke gunstige resultaten uit de fase 2-studies leidden tot hooggespannen verwachtingen voor de fase 3-trials naar de anti-CD3-antilichamen otelixizumab en teplizumab. De teleurstelling was groot toen er bij geen van beide trials begin 2011 een verschil in de primaire uitkomstmaat was tussen de interventie- en controlegroep [258]. In het onderzoek naar otelixizumab gold gestimuleerd C-peptide als maat voor de functionele bètacelmassa. De trial met teplizumab gebruikte als uitkomstmaten de verandering van de HbA1c-waarde en het percentage patiënten met HbA1c

<6,5% bij een insulinegebruik <0,5 E/kg per dag, beide 1 jaar na het begin van de therapie gemeten.

Details van de fase 3-trial met otelixizumab zijn nog niet gepubliceerd, maar een mogelijke verklaring voor het falen is dat de medicatiedosis lager was dan in de fase 2-trial, namelijk 25 mg in plaats van 48 mg. De dosis was gereduceerd in verband met de kans op reactivatie van het epstein-barrvirus. Ook in de teplizumab-studie was de dosis in de maximaal behandelde groep verlaagd tot 50% van de dosis in de fase 2-studie [114,258].

De teplizumab-studie onderstreept dat de keuze van primaire uitkomstmaten essentieel is. De keuze voor HbA1c-waarden als primaire uitkomstmaat in de fase 3-trial met teplizumab was opmerkelijk, aangezien de fase 2-studies geen duidelijk verschil in HbA1c-waarden tussen interventie- en controlegroep hadden laten zien. Ook is 1 jaar na de diagnose een insulinedosis van <0,5 E/kg per dag niet ongebruikelijk, waardoor de gelegenheid een verschil te meten beperkt werd.

Uiteindelijk bereikte 20% van zowel de behandelde groep als placebogroep de streefwaarden.

Post-hoc-analyse met een andere uitkomstmaat, namelijk HbA1c-streefwaarden volgens de reguliere behandelrichtlijnen (<7%) bij een stringentere insulinedosis (<0,25 E/kg per dag), toonde wél verschillen tussen de behandelde groep en de placebogroep. Er was bovendien een effect op het behoud van de bètacelfunctie (de uitkomstmaat in de fase 2-studie). Bijwerkingen bleven voornamelijk beperkt tot hoofdpijn en huidreacties. In beide groepen werden evenveel ernstige bijwerkingen gemeld. Hoewel de studiedoelen niet werden bereikt, lijkt het voorbarig om anti- CD3-antilichamen te diskwalificeren als potentiële therapie voor patiënten met recent gediagnosticeerde diabetes mellitus type 1.

VACCINATIE

Tegelijkertijd werden alternatieve therapeutische strategieën ontwikkeld en getoetst om resterende bètacellen te beschermen tegen auto-immuundestructie.

Eén zo’n strategie beoogt het herstel van specifieke regulatoire mechanismen van het immuunsysteem zelf. Ontspoorde afweerreacties kunnen immers door

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Chapter 2 Immune Intervention Trials towards a Cure for Type 1 Diabetes

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vooruitzichten van intensieve insulinetherapie. Het verdient overweging onderzoek naar immunotherapie ook bij kinderen uit te voeren, aangezien de helft van de nieuwe patiënten met diabetes mellitus type 1 jonger is dan 18 jaar en zij degenen zijn met het hoogste risico op complicaties door de ziekte op de lange termijn.

CONCLUSIE

De weg naar genezing van patiënten met diabetes mellitus type 1 is niet zonder hindernissen en terugslag, maar men boekt desondanks gestaag resultaten.

Genezing heeft al eens plaatsgevonden, zij het met forse bijwerkingen en risico’s.

Minder risicovolle, weefselspecifieke interventies en combinatietherapieën worden momenteel ontwikkeld en getoetst. Daarnaast zal verbetering van insulinepompen en -sensoren en eventueel directe koppeling van beide de behandeling en daarmee wellicht de kwaliteit van leven van patiënten met diabetes mellitus type 1 de komende jaren verbeteren. Voor nieuwe patiënten met diabetes mellitus type 1 zal het behoud van bètacelfunctie centraal komen te staan door nieuwe behandelingen die ingrijpen in de pathofysiologie van diabetes mellitus type 1.

LEERPUNTEN

● Diabetes mellitus type 1 is het gevolg van destructie van insulineproducerende ● bètacellen door auto-immuun T-cellen; hierbij ontstaan meestal ook auto- antilichamen;

● Immunotherapie bij patiënten met recent gediagnosticeerde diabetes mellitus ● type 1 zou het auto-immuunproces kunnen stoppen, zodat een deel van de bètacellen behouden blijft;

● De helft van een groep patiënten met diabetes mellitus type 1 die autologe ● stamceltransplantatie hadden ondergaan, hoefde 2 jaar later nog steeds geen insuline te gebruiken;

● Enkele minder ingrijpende immunotherapieën, zoals biologicals en vaccinaties, ● hadden tot nu toe geen significant effect in fase 3-studies bij patiënten met diabetes mellitus type 1;

● Bij onderzoek naar immunotherapie voor diabetes mellitus type 1 moet men ● rekening houden met heterogeniteit in patiëntengroepen; ook is er behoefte aan goede uitkomstmaten voor effectiviteit en biomarkers voor ziekteprogressie.

van de behandeling met insuline weerspiegelt. Een hogere HbA1c-waarde in de placebogroep zou dus kunnen duiden op inferieure insulinetoediening.

Aangezien het niet haalbaar is harde uitkomstmaten te hanteren, zoals het voorkómen van complicaties of mortaliteit en de kwaliteit van leven op lange termijn, is het handhaven van de C-peptideconcentratie – gemeten na stimulatie van de insulineafgifte – momenteel waarschijnlijk de geschiktste klinische uitkomstmaat.

Deze waarde verandert namelijk snel in de eerste jaren na het stellen van de diagnose ‘diabetes mellitus type 1’ en correleert ook sterk met complicaties op lange termijn [269].

HETEROGENITEIT IN RESPONS

De resultaten van de trials met immunotherapieën wijzen op opmerkelijke heterogeniteit in ziekte en in de respons op de behandeling, deels door etnische en genetische verschillen. In de fase 3-studie naar teplizumab was bescherming van de bètacelfunctie meer uitgesproken bij kinderen (8-11 jaar) en bij patiënten geïncludeerd in de VS. Ook viel op dat de afname van C-peptide bij patiënten in India, Canada en Mexico veel kleiner was dan bij patiënten geïncludeerd in Europa, Israël of de VS, ongeacht therapie. In de abatacept-trial stond het handhaven van de C-peptideconcentratie in de complete trial in scherp contrast met een significante daling van deze waarde in ‘non-white’ deelnemers [198,238,258].

Recente studies leveren interessante biomarkers voor therapeutische effectiviteit en selectie van patiënten [241]. Zo blijkt de concentratie C-peptide na stimulatie een belangrijke klinische voorspeller van effectiviteit van immunotherapie en is het ontbreken van een auto-immuunrespons vóór transplantatie voorspellend voor succes bij een eilandtransplantatie [124]. Deze inzichten zijn gezamenlijk een opstap naar therapie op maat.

BIJWERKINGEN

In de meeste trials met individuele immuunsuppressiva lijkt behandeling vooral een initiële vertraging in het verlies van bètacelfunctie te bereiken, waarna de concentratie C-peptide na stimulatie enkele maanden later in de behandelde én onbehandelde groep parallel daalt. Hoewel dit een beperkt resultaat lijkt, zijn de bijwerkingen in deze studies ook beperkt, in tegenstelling tot de risico’s van de effectievere autologe stamceltherapie. Een combinatie van verschillende immuunsuppressiva, eventueel met tolerantie-inducerende therapie, zou uitkomst kunnen bieden bij de tekortkomingen van de individuele therapieën. Desondanks moet gewaakt worden voor subtherapeutische doseringen die paradoxaal versnelde ziekteprogressie zouden kunnen geven, zoals mogelijk bij lage doses van teplizumab gebeurd is [258].

Ernstigere bijwerkingen kunnen, als zij tijdelijk en omkeerbaar zijn, wellicht opwegen tegen het risico op verminderde kwaliteit van leven dat patiënten op de lange termijn lopen, ondanks insulinetherapie [15]. Voor elke nieuwe studie zullen de risico’s en baten opnieuw afgewogen moeten worden en afgezet tegen de steeds betere

3

Fate of Transplanted Islets,

Local Insights

Cell Transplantation 2017;26(1):1-9

Cornelis R. van der Torren, Jessica S. Suwandi, DaHae Lee, Ernst-Jan T. van ’t Wout, Gaby Duinkerken, Godelieve Swings, Arend Mulder, Frans H.J. Claas, Zhidong Ling, Pieter Gillard, Bart Keymeulen, Peter in ’t Veld and Bart O. Roep

Identification of Donor Origin and Condition of Transplanted Islets in situ in the Liver of a Type 1 Diabetic Recipient

Chapter 3

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Chapter 3 Fate of Transplanted Islets, Local Insights

3

INTRODUCTION

Islet transplantation is an effective treatment for brittle type 1 diabetes and it allows most patients to achieve insulin independence. Transplanted beta-cell mass is an important determinant of transplantation success. Single donor transplantation are preferred, but islets from multiple donor organs and repeated transplantations are often required to achieve optimal function [135]. Although multi-donor transplantation has improved transplantation outcome, it complicates understanding of improvements in isolation, transplantation and immunosuppressive strategies.

Identifying the fate of individual donor grafts is necessary to interpret changes in outcome with novel transplantation strategies. We previously reported on donor- specific alloreactive responses and recurrent autoimmunity in multi-donor islet transplants by investigating circulating immune cells [118,124,127,234]. However, it remains to be determined how immunity measured in peripheral blood relates to local immunity in islet transplantation.

Opportunities to investigate transplanted islets in situ are rare. Percutaneous techniques have reduced side effects of islet transplantation, while investigating an intraportal graft by transcutaneous liver biopsy has proven infeasible [287]. Risk of complications precludes repeated liver biopsies or surgical major biopsies to access transplanted islets. Therefore, in situ studies can only be performed post mortem or on incidental patients who would require liver surgery. Identification of islet material in situ is necessary to investigate donor specific effects. Donor and recipient human leukocyte antigen (HLA) typing are usually known and differ in unmatched cases. We previously established a bank of human HLA specific monoclonal antibodies to study humoral rejection in transplantation [298]. The ubiquitous expression of HLA-class I would allow for employment of these antibodies to differentiate between recipient and individual donors by immunohistochemistry.

We investigated islet donor origin in the case of a 61-year old woman treated with islet transplantation for her brittle type 1 diabetes, who died of cerebral hemorrhage four months after receiving two intraportal islet grafts. Immunosuppression consisted of anti-thymoglobulin and methylprednisolone induction therapy and tacrolimus and mycophenolate mofetil maintenance therapy. She received islets from four donors in the first transplantation and from two donors in a second transplantation after six weeks. All donors had complete HLA-A, -B and –DR mismatch with the recipient. At time of death she had a functioning graft with non-fasting C-peptide of 2.02 ng/ml at 220 mg/dl glycemia while using 13 units of insulin per day. Auto- and allo-reactive immune responses of T-cells and antibodies were monitored per protocol before and after transplantation.

ABSTRACT

Transplantation of islet allografts into type 1 diabetic recipients usually requires multiple pancreas donors to achieve insulin independence. This adds to the challenges of immunological monitoring of islet transplantation currently relying on surrogate immune markers in peripheral blood. We investigated donor origin and infiltration of islets transplanted in the liver of a patient with type 1 diabetes who died of hemorrhagic stroke four months after successful transplantation with two intraportal islet grafts combining six donors.

Immunohistological staining for donor HLA using a unique panel of human monoclonal HLA-specific alloantibodies was performed on liver cryosections after validation on cryopreserved kidney, liver and pancreas and compared with auto- and alloreactive T-cell immunity in peripheral blood.

HLA specific staining intensity and signal-to-noise ratio varied between tissues from very strong on kidney glomeruli, less in liver, kidney tubuli and endocrine pancreas to least in exocrine pancreas, complicating the staining of inflamed islets in an HLA- disparate liver. Nonetheless, five islets from different liver lobes could be attributed to donors 1, 2 and 5 by staining patterns with multiple HLA-types. All islets showed infiltration with CD8⁺ cytotoxic T-cells that was mirrored by progressive alloreactive responses in peripheral blood mononuclear cells to donors 1, 2 and 5 after transplantation. Stably low rates of peripheral islet autoreactive T-cell responses after islet infusion fit with a complete HLA mismatch between grafts and recipient and exclude the possibility that the islet infiltrating CD8 T-cells were autoreactive.

HLA-specific immunohistochemistry can identify donor origin in situ and differentiate graft dysfunction and immunological destruction.

Acknowledgements

Authors thank Dr. John Scharenberg of Sanquin Blood Supply for AlexaFluor 488 labelling of human monoclonal antibodies.

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3

50,000 irradiated (3000 rad) stimulator cells per well at 37°C/5% CO₂. After 5 days,

3H-thymidine (1.0 μCi per well) was added and ³H-thymidine incorporation was measured on a beta-plate counter (Wallac-LKB Betaplate 1205, PerkinElmer Wallac, Turku, Finland) after 16 hours. Proliferation in response to phytohaemagglutinin was used as positive control. Results were interpreted as SI compared to background value (responder only + stimulator only). Production of different cytokines was measured with Luminex technology using a human Th1/Th2 Bio-plex cytokine kit (Bio-Rad, Veenendaal, the Netherlands), including IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ) and tumor necrosis factor (TNF), according to the manufacturer’s protocol.

Screening for the presence of HLA-class I and class II-specific antibodies was performed on all available samples by enzyme-linked immunosorbent assay (ELISA, LAT class I&II, One Lambda, Canoga Park, CA, USA). Islet cell autoantibodies (ICA), autoantibodies against IA-2 protein (IA-2A) and GAD, were measured as described before [65]. Briefly, ICA were determined by indirect immunofluorescence. IA-2A and GAD were determined by liquid phase radiobinding assays.

HLA-specific antibody staining

Human monoclonal antibodies specific for one or more HLA-subtypes were selected from a previously described panel [185,298]. In short, heterohybridomas were created by Epstein-Barr virus transformation and cloning of B-lymphocytes of multiparous women. The HLA-specificities of the produced human monoclonal antibodies were validated using a complement-dependent cytotoxicity test against PBMC. HLA specificities were confirmed by flow cytometry on single HLA antigen expressing cell lines [322] and on single antigen beads [166]. Five antibodies were selected that could collectively differentiate between the six islet donors on HLA- typing (Table 3.1 & 3.2). Antibodies were directly labelled with Alexa Fluor 488 (AF488) by Sanquin Blood Bank (Leiden, The Netherlands). Cryopreserved tissue was cut in consecutive 5 µm sections on a cryotome (Thermo Fisher Scientific, Waltham, MA, USA), air-dried and fixed in 4ºC acetone for 10 minutes. Sections were stained overnight at 4ºC with AF488 labelled HLA-specific antibodies (1:100).

Then, sections were stained with 4’,6-diamidino-2-phenylindole (DAPI, 1 ng/ml;

(Sigma-Aldrich, Zwijndrecht, The Netherlands)) and mounted with Mowiol (Sigma- Aldrich). For enhanced and multifluorescence staining, primary staining was followed by combination of rabbit-anti-Alexa488 (Invitrogen, Bleiswijk, The Netherlands), guinea pig-anti-insulin (polyclonal immunoglobulin G (IgG), in house), rabbit-anti- glucagon (polyclonal IgG, in house), mouse-anti-CD45 (Dako, Heverlee, Belgium) and/or mouse-anti-CD8 (NovaCastra, Rijswijk, The Netherlands) overnight at 4ºC and, thereafter, anti-rabbit-Cy3 (Dako), anti-guinea pig-Alexa 647 (Dako) and/or anti- mouse-AF488 (Dako), as applicable. These slides were mounted with DAPI (0.12 ng/ml) in fluorescence mounting medium (Dako). Positive HLA antibody staining was assessed by comparing fluorescence on insulin positive cells between islets in the same section and between HLA antibodies staining the same islet in consecutive sections and cells were counted manually.

MATERIALS AND METHODS

Blood samples were collected in sodium heparin tubes and serum tubes (BD vacutainer, Breda, the Netherlands) containing silicate granulate for immune monitoring before and at 4, 6, 9 and 12 weeks after transplantation with signed informed consent of the patient and according to the approved protocol [124].

Autopsies and studies on organ specimens were performed after obtaining oral informed consent from the patient’s family. For antibody optimization, cryopreserved kidney, liver and pancreas tissue was obtained from left over specimen selected to match allo-antibody HLA specificity. All material was treated according to local and institutional regulations with approval from the Medical Ethical Committee of the Free University Brussels, Belgium and in accordance with the 2008 revised principles of the Declaration of Helsinki.

Peripheral blood immune analysis

Autoimmune responses were determined blinded from clinical results. Lymphocyte stimulations test was performed before and at regular intervals after transplantation and on lymphocytes derived from different organs upon autopsy, as described before [242]. Briefly, 150,000 fresh peripheral blood mononuclear cells (PBMC) per well or tissue derived lymphocytes per well were cultured in 96-well round-bottom plates in Iscove’s Modified Dulbecco’s Medium with 2 mmol/l glutamine (Gibco, Paisley, Scotland) and 10% pooled human serum in the presence of antigen, interleukin-2 (IL-2, 35 U/ml; Novartis, Arnhem, The Netherlands) or medium alone in triplicates.

After 5 days ³H-thymidine (0.5 μCi per well; DuPont NEN, Boston, MA) was added and ³H-thymidine incorporation was measured after 16 hours on a beta-plate counter. Antigens analyzed included islet autoantibody-2 (IA-2, 10 μg/ml), glutamate decarboxylase-65 (GAD65, 10 μg/ml), insulin (25 μg/ml) and tetanus toxoid (‘recall’

antigen, 1.5 LF/ml). Results were interpreted as stimulation index (SI) compared to medium value and with a cutoff value of SI >3.

Alloreactive T-cell responses were determined by cytotoxic T-lymphocyte precursor (CTLp) assay and mixed lymphocyte reaction (MLR). The CTLp assay to determine cytotoxic T-cell alloreactivity was described before[34]. Briefly, PBMC were cultured in a limiting dilution assay (40,000 to 625 cells/well, 24 wells per concentration) with different irradiated stimulator PBMC expressing HLA-class I antigens matching beta-cell grafts (50,000 cells/well). Cells were cultured for 7 days at 37°C in 96-well round-bottom plates in RPMI1640 medium (Gibco BRL, Paisely, UK) with 3 mmol/l L-glutamine, 20 U/ml IL-2 and 10% pooled human serum. Then, Europium-labelled (Fluka, Buchs, Switzerland) graft HLA-specific target cells (5,000 cells/well) were added for 4 hours. Wells were scored positive if the Europium release through target cell lysis exceeded spontaneous release +3x standard deviation. Quantification of CTLp frequencies was performed by computer software [274]. In parallel, one- way mixed lymphocyte cultures were set up in triplicates in 96-well V-bottom plates (Costar, Cambridge, MA, USA) in 150 μl RPMI with 2 mmol/l L-glutamine (Gibco) and 10% pooled human serum. Responder cells (40,000) were incubated with

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Chapter 3 Fate of Transplanted Islets, Local Insights

3

0 100 200 300 400 500

CTLpfrequency/106PBMC

C A

0 500 1000 1500 2000 2500

0 10 20 30

MMFdose(mg/day) TACtroughlevels(ng/ml)

B

0 1 2 3 4

PlasmaC-peptide(ng/ml)

0 10 20 30 40 50

Proliferation(SI)

D

0 10 20 30 40 50

IFNγ/IL10ratio

E

Weeks after transplantation

Tx2 1, 2, 3, 4

2, 3, 4 1, 3, 4, 5, 6

1, 4, 5, 6 1, 3, 4, 5, 6

6 Donor match

x x Tx2x x

Tx2

Tx2

10 15

Tx2

0 5

Figure 3.1. Immune suppression, graft function and alloreactive T-cell responses monitored before and after transplantation.

A: Timing of immune suppression: thymoglobulin (horizontal bar), methylprednisolone pulses (x), mycophenolate mofetil daily dose (dashed line) and tacrolimus trough levels (squares).

B: Random C-peptide measurements. C:

Alloreactive cytotoxic T-cell precursor (CTLp) frequency to 6 human leukocyte antigen (HLA) mismatched stimulator target combinations was measured. Reactivity 8 weeks after transplantation (filled triangle and square) suggest reactivity to HLA A11 (donor 1&5) and/

or B62 (donor 1, 3, 5 & 6). After 12 weeks, reactivity to stimulators depicted with open circles and diamonds suggest HLA A3 or B7 (both donor 2&4) reactivity, although A1, A26, B8 and/or B60 reactivity (donor 1, 2 and/or 3) is also possible. Further, changing reactivity to filled symbols suggest upcoming HLA B44 reactivity matching donor 1. D: Alloreactive T-helper proliferation was suppressed after transplantation. E: Mixed lymphocyte reaction (MLR) cytokine response showed increasing interleukin-10 (IL-10) production, but stable interferon gamma (IFNγ) response leading to sharp decrease in IFNγ/IL-10 ratio. Symbols represent matched stimulators. Tx2: second transplant; MMF: mycophenolate mofetil;

TAC: tacrolimus; PBMC: peripheral blood mononuclear cells; SI: stimulation index.

RESULTS

Immune response analysis

Immune responses were determined in peripheral blood before and after transplantation. Autoreactive T-cell responses were tested by lymphocyte stimulation test to autoantigens and showed response to GAD65 (SI 13.2) before transplantation, but very low autoantigen specific responses were shown after transplantation.

Insulin autoantibodies were present before transplantation and remained unchanged thereafter. Alloreactive cytotoxic T-cell frequency was reduced in the first 6 weeks after transplantation. After 8 weeks, responses increased to HLA-A11 (donors 1 and 5) and/or HLA-B62 of (donors 1, 3, 5 and 6). By week 12 additional responses emerged to donor 1 (HLA-B44) and donor 2, 3 and/or 4 (HLA-A1, A3, A26, B7, B8 and/or HLA-B60) (Figure 3.1). Alloreactive T-helper cell responses in MLR decreased after transplantation correlating with increased IL-10 levels and reduced IFNγ/IL-10 ratio (Figure 3.1). No alloreactive antibodies were measured before or after treatment (Tx).

Islet Lobe BRO11F6

(D1,2,5) JOK3H5

(D1,2,6) SN230G6

(D4,5,6) BVK1F9

(D3) DK7C11

(D1) Conclusion

1 LL + - + - - D5

2 LR + ± - - - D2

3 LR + + - - + D1

4 LR + ± + - ± D5

5 LR + - + - - D5

Table 3.1. Islet donor identification by HLA antibodies. ± = inconclusive. D: donor; LL: left liver lobe;

LR: right liver lobe.

Donor Antibodies

HLA-A HLA-B BRO11F6 JOK3H5 SN230G6 BVK1F9 DK7C11

1 A10 (26)

A11 B12 (44)

B15 (62) A11 B12 -- -- B12

2 A3

A19 (30) B7

B40 (60) A3 B40 -- -- --

3 A1

A10 (26) B8

B15 (62) -- -- -- B8 --

4 A2 B7

B17 (57) -- -- A2 / B17 -- --

5 A2

A11 B15 (62)

B27 A11 -- A2 -- --

6 A2

A19 (29) B13

B15 (62) -- B13 A2 -- --

Patient A24 B18 A24 -- -- -- --

Table 3.2. Donor and HLA antibody staining combinations.