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LINKÖPING UNIVERSITY MEDICAL DISSERTATIONS

NO 1277

Combinations of type 1 diabetes, celiac disease and allergy -

An immunological challenge

Anna Kivling

Division of Pediatrics

Department of Clinical and Experimental Medicine

Faculty of Health Sciences, Linköping University

SE-581 85 Linköping

Linköping 2011

©Anna Kivling

Cover design by Anna and Helle Kivling.

ISBN: 978-91-7393-021-5

ISSN 0345-0082

Ownership of copyright for paper I and III remains with the authors.

Paper I was originally published by John Wiley & Sons, Inc.

Paper III was originally published by Elsevier BV.

Paper IV has been reprinted with kind permission from John Wiley & Sons, Inc.

During the course of the research underlying this thesis, Anna Kivling was enrolled in

Forum Scientium, a multidisciplinary doctoral programme at Linköping University,

Sweden.

Printed by LiU-tryck, Linköping 2011

What if everything around you

Isn't quite as it seems?

What if all the world you think you know

Is an elaborate dream?

And if you look at your reflection

Is it all you want it to be?

What if you could look right through the cracks?

Would you find yourself

Find yourself afraid to see?

Right where it belongs, Trent Reznor

NiN album With teeth, 2005

To me, myself, and I. With love.

Abstract

The immune system is composed of a complex network of different cell types

protecting the body against various possible threats. Among these cells are T-helper

(Th) cells type 1 (Th1) and type 2 (Th2), as well as T regulatory (Treg) cells. Th1 and Th2

are supposed to be in balance with each other, while Tregs regulate the immune

response, by halting it when the desired effect, i.e. destroying the threat, is acquired.

However, sometimes this intricate interplay in the immune system is disturbed,

leading to diseases as type 1 diabetes (T1D), celiac disease or allergic disease. According

to the paradigm claiming that Th1- and Th2-cells inhibit each other a coexistence of a

Th1-deviated disease and a Th2-deviated disease seems unlikely.

This thesis aimed to examine the immune response with focus on subsets of T-cells in

children with T1D, celiac disease, allergy, or a combination of two of these diseases, in

comparison to reference children (healthy).

In line with previous findings we observed that children with celiac disease showed a

decreased spontaneous Th2-associated secretion, whereas children with allergic

disease showed increased birch- and cat-induced Th2-associated response.

The most remarkable results in this thesis are those observed in children with

combinations of diseases. The combination of T1D and celiac disease decreased the

Th1-associated response against several antigens, but instead displayed a more

pronounced Treg-associated response. Further, in children with combined T1D and

allergy an increased Th1- and Th2-associated response was seen to a general stimulus,

and an increased birch-induced Th1-, Th2-, Treg- and pro-inflammatory response. In

contrast, the combination of allergy and celiac disease showed a decreased

spontaneous Th1-, Th2-, Treg- and pro-inflammatory response.

In conclusion, we observed that two Th1-deviated diseases in combination suppress the

immune response and increase the regulatory activity. Further it seems that allergy has

the ability to shift the immune response in diverging directions depending on which

disease it is combined with. The observed suppressive effect might be due to

exhaustion of the immune system from the massive pressure of two immunological

diseases in combination, while the pronounced Treg response might be caused by an

attempt to compensate for the dysfunction. These results shed some light on the

intriguing and challenging network that constitutes the immune system, and hopefully

give clues regarding disease prevention and treatment.

Populärvetenskaplig sammanfattning

I immunförsvaret finns ett flertal olika celltyper som samarbetar för att skydda

kroppen mot potentiellt skadliga ämnen. Bland dessa finns T-hjälpar celler (Th) av typ

1 och 2, vars uppgift är att hjälpa andra celler att utföra sina uppgifter. Th1- och Th2-

celler tros vara i balans med varandra, och hämmar därför vid behov varandra. De

reglerande T-cellerna (Treg) har istället en reglerande effekt med uppgift att bromsa

immunförsvaret i tid för att undvika skada på kroppens egna celler. Ibland störs

balansen, vilket kan leda till olika sjukdomar, som de autoimmuna sjukdomarna typ 1-

diabetes (T1D) och celiaki men också uppkomst av allergi. Givet den balans som råder

mellan Th1 och Th2, borde det vara ovanligt med att en sjukdom med Th1-inslag ska

kunna existera tillsammans med en med Th2-inslag, men denna kombination av

sjukdomar är inte ovanligare än vad som är förväntat i befolkningen.

Denna avhandling syftar till att studera immunsvaret med fokus på olika undergrupper

av T-celler (bla Th1, Th2 och Treg), hos barn med T1D, celiaki och allergi, samt även

kombinationer av två av dessa sjukdomar, men även jämfört med friska barn .

Vi fann att barn med celiaki, en sjukdom med Th1-inslag, enligt förväntan reagerade

med ett lågt Th2-svar, och att barn med allergi, en sjukdom med Th2-inslag, reagerade

starkt mot både björk och katt, också det enligt våra förväntningar.

Immunförsvaret hos barn med kombinationer av dessa immunologiska sjukdomar har

inte utforskats i någon stor utsträckning, och der är här vi fann de mest oväntade

resultaten. Vi fann att barn med kombinationen av T1D och celiaki uppvisar både ett

minskat Th1-svar mot ett flertal ämnen, samt ett mer uttalat Treg-svar. Barn med

allergi verkar också ha förmågan att förskjuta immunförsvaret i olika riktningar

beroende på vilken annan sjukdom de kombineras med. Kombinationen av T1D och

allergi ger upphov till ett ökat Th1, Th2 samt Treg-svar mot björk, medan

kombinationen av celiaki och allergi istället ger upphov till ett minskat spontant Th1-,

Th2- samt Treg-svar.

Sammanfattningsvis tyder våra resultat på att två sjukdomar med Th1-inslag trycker

ned immunförsvaret, men ökar den reglerande aktiviteten. Dessutom verkar allergi

kunna förskjuta immunförsvaret i olika riktningar beroende på vilken annan sjukdom

den kombineras med. Det nedtryckta svar vi ser kan bero på att immunförsvaret är

utmattat från det tryck som är följden av att bära på har två immunologiska

sjukdomar. Den mer uttalade reglerande effekten kan dessutom komma sig av att

immunförsvaret försöker kompensera för den störda balansen. Förhoppningsvis kan

dessa resultat sprida lite ljus över det intrikata nätverk immunförsvaret består av samt

även ha effekt på prevention och behandling.

Contents

Original Publications ................................................................................................. 5

Abbrevations .............................................................................................................. 7

Review of the literature ........................................................................................... 13

Introduction to the immune system ................................................................... 13

T-helper cells .................................................................................................... 13

Disturbance in Th1-Th2-Treg balance ............................................................ 15

Regulatory T-cells ............................................................................................ 15

Cytokines and chemokines .............................................................................. 18

Type 1 diabetes ......................................................................................................... 19

Pathogenesis and epidemiology .......................................................................... 19

Immunology ........................................................................................................ 20

Celiac disease ........................................................................................................... 22


Immunology .........................................................................................................24

Allergy....................................................................................................................... 27


Immunology ........................................................................................................ 28

Combinations of Type 1 diabetes, Celiac disease and Allergy ............................... 30

Combination of type 1 diabetes and celiac disease ............................................ 30

Combination of type 1 diabetes and allergy ....................................................... 31

Children with combination of celiac disease and allergy .................................. 31

Cryopreservation ...................................................................................................... 33

Aims of the thesis .................................................................................................... 35

Material and methods ............................................................................................. 37

Study population .................................................................................................. 37

Paper 1 ............................................................................................................... 38

Paper II ............................................................................................................. 39

Paper III ............................................................................................................ 39

Paper IV ........................................................................................................... 40

Paper V............................................................................................................. 40

Diagnostic criteria ............................................................................................ 41

Laboratory analyses .............................................................................................42

Isolation and in vitro stimulation of peripheral blood mononuclear cells ..42

Freezing, thawing and in vitro stimulation of PBMC (paper III) .................. 43

Collection of PBMC and cell supernatants (paper II, III and IV) .................. 45

Enzyme linked immunospot (ELIspot) (paper I) ........................................... 45

Multiplex fluorochrome technique (Luminex) (paper II) .............................47

Real-time reversed-transcriptase polymerase chain reaction (RT-PCR)

(paper III and IV) .............................................................................................50

Enzyme linked immunosorbent assay (ELISA) (paper III) ............................ 52

Flow cytometry ................................................................................................. 52

Statistical analyses ........................................................................................... 54

Ethics ................................................................................................................ 55

Results and Discussion ............................................................................................ 57

Children with T1D showed a decreased spontaneous IL-15 secretion and a

decreased spontaneous and PHA-induced expression of FOXP3 ..................... 57

Children with celiac disease showed decreased spontaneous IL-4 and IL-15

secretion but an increased expression of spontaneous, insulin-, gluten- and

PHA-induced FOXP3 ........................................................................................... 61

Children with allergy showed an increased birch- and cat-induced IL-4

secretion, an increased spontaneous IFN-γ, IL-10 and IL-12 secretion .............65

Children with a combination of T1D and celiac disease showed a decreased

inhalant- and food-antigen-induced IFN-γ secretion, however an increased

Treg-associated response ................................................................................... 70

Children with a combination of T1D and allergy showed an increased PHA-

induced IFN-γ and IL-4 secretion, as well as an increased birch-induced

secretion of several cytokines ..............................................................................74

Children with a combination of celiac disease and allergy showed a decreased

spontaneous secretion of several cytokines ...................................................... 78

Methodological data ........................................................................................... 80

Summary and Conclusion ...................................................................................... 86

Future perspectives ................................................................................................. 89

Acknowledgement ................................................................................................... 91

References ............................................................................................................... 95

~ 5~

ORIGINAL PUBLICATIONS

This thesis is based on the following papers, which will be referred to in the text by

their Roman numerals.

I: Combinations of common paediatric diseases deviate the immune response in

diverging directions

Nilsson L, Kivling A, Jalmelid M, Fälth-Magnusson K, Faresjö M

Clinical and Experimental Immunology. 2006; 146: 433-42

II: Diverging immune responses when allergy, type 1 diabetes and celiac disease coexist

Kivling A, Nilsson L, Åkesson K, Fälth-Magnusson K, Faresjö M

Manuscript

III: How and when to pick up the best signals from markers associated with T-

regulatory cells?

Kivling A, Nilsson L, Faresjö M

Journal of Immunological Methods, 2009; 345: 29-39

IV: Diverse foxp3 expression in children with type 1 diabetes and celiac disease

Kivling A, Nilsson M, Fälth-Magnusson K, Söllvander S, Johansson C, Faresjö M

Annals of the New York Academy of Sciences. 2008; 1150;273-277

V: Combined type 1 diabetes and celiac disease in children give raise to a more

pronounced Treg population

Kivling A, Åkesson K, Nilsson L, Faresjö M

Manuscript

~ 7 ~

ABBREVIATIONS

AGA Anti-gliadin antibody

AP Alkaline phosphatase

APC Allophycocyanin

BAL Bronchoalveolar lavage

βLG β-lactoglobulin

BSA Bovine serum albumin

cDNA Complementary DNA

Ct Cycle threshold

CTLA-4 Cytotoxic T-lymphocyte antigen-4

Cy Cyanin

DC Dendritic cells

DMSO Dimethyl sulfoxide

dNTP Deoxyribonucleotide triphosphate

ELISA Enzyme linked immunosorbent assay

ELIspot Enzyme linked immunospot

EMA Endomysial antibody

ESPGAN The European Society of Paediatric Gastroenterology and Nutrition

ESPGHAN The European Society of Paediatric Gastroenterology, hepatology

and Nutrition

~ 8 ~

FCS Fetal calf serum

FSC Forward scatter

FGF Fibro blast growth factor

FITC Fluorescein isothiocyanate

FOXP3 Forkhead box protein 3

GAD Glutamic acid decarboxylase

G-CSF Granulocyte colony-stimulating factor

GFD Gluten free diet

GM-CSF Granulocyte macrophage colony-stimulating factor

HbA1c Hemoglobin A1c

HCl Hydrochloric acid

HLA Human leukocyte antigen

IA-2 Thyrosinphosphatase

IEL Intraepithelial lymphocytes

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IL-1ra IL-1 receptor antagonist

IL-2RA IL-2 receptor alpha

IMDM Iscove´s modification of Dulbeccos medium

ISAAC International Study of Asthma and Allergies in Childhood

~ 9~

LPS Lipopolysaccharide

MFI Median fluorescence intensity

mRNA Messenger RNA

NK Natural killer

NOD Non-obese diabetic

nTreg Naturally occurring regulatory T-cell

OD Optic density

OVA Ovalbumin

PBMC Peripheral blood mononuclear cells

PBS Phosphate buffered saline

PDGF Platelet-derived growth factor

PE Phycoerythrin

PerCP Peridinin-chlorophyll proteins

PHA Phytohaemagglutinin

PTPN22 Protein tyrosine phosphatase, non-receptor type 22

rRNA Ribosomal RNA

RT Room temperature

RT-PCR Reversed transcriptase polymerase chain reaction

sCTLA-4 Soluble cytotoxic T-lymphocyte antigen-4

SIT Specific immunotherapy

SPT Skin prick test

~ 10~

SSC Side scatter

T1D Type 1 diabetes

Tc Cytotoxic T-cell

TGF Tumor Growth Factor

Th T-helper

TNF Tumor necrosis factor

TR1 T-regulatory cells 1

Treg Regulatory T-cell

TT Tetanus toxoid

tTG Tissue transglutaminse

tTGA Tissue translutaminase antibody

VEGF Vascular endothelial growth factor

ZnT8 Zinc transporter 8

~ 11~

CHEMOKINE NOMENCLATURE

The four different groups of chemokines are C-X-C, C-C, C and C-X3-C,

indicating number and spacing of conserved cysteines [1]. The letter “L”

indicates a ligand whereas the letter “R” indicates receptor[2].

CXCL9=MIG

CXCL10=IP-10

CXCL11=I-TAC

CCL3=MIP1α

CCL4=MIP1β

CCL5=RANTES

CCL7=MCP-3

CCL11=Eotaxin

CCL2=MCP-1

CCL17=TARC

CCL1=I-309

CXCL8=IL-8

~ 13~

REVIEW OF THE LITERATURE

Introduction to the immune system

The word immunity comes from the Latin word “immunis”, meaning exempt.

The immunological science is believed to originate from Jenner who discovered

that cowpox could be used as vaccine against human smallpox; however it was

not until late 19th century that microorganisms were identified as the cause for

infections. The function of the immune system is to protect the body from any

foreign, and possibly harmful invader, like viruses, bacteria, fungi, parasites and

tumors [3]. The immune response is generally divided into innate and adaptive

immunity. The innate immunity, or non-specific, is the first line of defense,

including physical barriers as well as monocytes, dendritic cells (DC),

granulocytes, macrophages and natural killer (NK) cells. Adaptive immunity

consists of highly specialized lymphocytes; T-helper (Th) cells, cytotoxic T-cells

(Tc) and B-cells. The adaptive immunity is long-lasting in contrast to the innate

immunity.

T-helper cells

Several cells cooperate in the immune system in order to protect the body from

and defeat possible harmful threats. In 1980´s Mossman et. al. [4] presented a

murine model describing two types of Th-cells, Th1 and Th2 respectively, with

different cytokine and chemokine patterns, and consequently different

functions. T-helper-1 cells secrete e.g. the cytokines interleukin (IL)-2, tumor

necrosis factor (TNF)-β and interferon (IFN)-γ and they are stimulated by IL-2,

IL-12, tumor growth factor (TGF)-β and IFN-γ (figure 1) [5, 6]. Additionally, Th1-

cells express chemokine receptor CXCR3 (receptor for CXCL9, CXCL10 and

~ 14~

CXCL11) and to some extent CCR5 (receptor for CCL3, CCL4 and CCL5) (figure 1)

[7]. In the immune response Th1-cells are involved in defense against

intracellular bacteria and viruses, as well as activation of cytotoxic and

inflammatory responses and delayed-type hyper-sensitivity reactions [5, 6].

T-helper-2 cells secrete e.g. IL-4, IL-5, IL-9 and IL-13, and are stimulated by IL-2

and in particular by IL-4 (figure 1) [5, 6]. The chemokine receptors CCR3

(receptor for CCL7 and CCL11), CCR4 (receptor for CCL2, CCL3, CCL5 and

CCL17) and CCR8 (receptor for CCL1) are all associated with a Th2-response

(figure 1) [7-9]. T-helper-2 cells are involved in antibody production, eosinophil

proliferation and function, as well as phagocyte-independent host defense

against for example helminths, a reaction mediated by immunoglobulin (Ig)E

and eosinophils [5, 6].

Cytokines associated with Th1-cells, e.g. IFN-γ, exert an inhibitory effect on Th2-

cells, and Th2-associated IL-4 inhibits Th1-development, a feature that gives rise

to a balance between Th1- and Th2-cells [10, 11].

This description is however a simplification of the intricate interplay between

different cells. Cells secreting IL-9 and small amounts IL-10 are termed Th9;

they are activated by IL-4 and TGF-β and have no suppressive functions but act

in a pro-inflammatory manner [12, 13]. Another subpopulation of Th-cells are

Th17 cells, which are induced by TGF-β, IL-1β, IL-6 and IL-23 [14]. T-helper-17

cells have a pro-inflammatory effect, involved in autoimmune disease as well as

in allergy and asthma and they secrete IL-6, IL-17A, IL-17B, IL-22 and CXCL-8

(reviewed in [15]).

~ 15~

Disturbance in Th1-Th2-Treg balance

It is widely accepted that a disturbance in the balance between Th1 and Th2

might lead to different diseases [5, 6, 16, 17]. A Th1-deviation is commonly

associated with autoimmune disease, among them are rheumatoid arthritis and

multiple sclerosis besides T1D and celiac disease, while Th2-deviation is

associated with measles virus infection, atopic disorders and in addition a

successful pregnancy and transplantation tolerance [5, 6, 16, 17]. However, as

there is no clear cut between the different cell populations, growing evidence

points out the need for an expansion of different subsets of lymphocytes [18-21].

Regulatory T-cells

The concept of a balance between Th1 and Th2, and two distinct separated sub-

populations has been a dogma for several years. This concept is today more and

more considered as a working model of the balance in the immune response

and not the one and only explanation. Already in 1970’s immunologists talked

about suppressive T-cells [22], and those cells were further studied until mid-

1980’s when studies regarding suppressive cells vanished due to draw-backs in

characterization of the suppressive cells [23]. During the 1990’s, however

suppressive cells re-entered the scientific scene, when Sakaguchi et. al. [24]

described CD4+ CD25+ cells with regulatory features in mice, further on they

were also described in humans [25]. These so called regulatory T-cells (Treg)

maintain immune homeostasis and self-tolerance [26]. Naturally occurring Treg

(nTreg) develop in thymus and are CD4+, constitutively express CD25, the α-

chain of the IL-2 receptor (figure 1) [24, 27]. Only approximately 1-2% of the

CD4+ CD25+ display the strongest regulatory function in humans, and this

population is usually termed CD4+ CD25high [28]. Several markers and cytokines

have been implied to be associated with Treg cells, both positively and

negatively, among them are forkhead box protein 3 (FOXP3), cytotoxic T-

~ 16~

lymphocyte antigen-4 (CTLA-4), CD39, CD45RA, CD127, IL-10 and TGF-β (figure

1) [25, 26, 29-31].

The transcription factor FOXP3 is a specific marker of Treg cells in mice, but not

in humans according to Walker et. al. [29]. They showed that FOXP3 could be

induced after activation of human CD4+ CD25- cells, and the up-regulated

FOXP3+ population acquired suppressive features after prolonged culture [29].

However, FOXP3 is one of the best available markers so far. FOXP3 has the

ability of directing the immune response through suppression of activation,

proliferation and effector functions, for example cytokine production, and has

been shown to control Treg development in mice [32]. The marker CTLA-4

(CD152) has been shown to be intracellularly expressed in CD4+ CD25+ cells,

remains up-regulated after activation and inhibits the activation of naïve T-cells

through down-regulation of CD80/CD86 [25, 33, 34]. An alternative spliced form

of CTLA-4 constitutes the soluble form (sCTLA-4), normally found in low levels

yet shown to be increased in several autoimmune diseases [35-37]. The

ectonucleotidase CD39 is expressed on FOXP3+ cells and suppresses

inflammation, hence an appropriate marker for Treg (figure 1) [30, 38].

Recently three different populations of cells expressing CD45RA and FOXP3 in

different proportions were identified; CD45RA+ FOXP3low was characteristic for

resting Treg cells, while activated Treg cells expressed CD45RA- FOXP3high and

CD45RA- FOXP3low cells were expressed on non-suppressive Treg cells, making

CD45RA a negatively Treg associated marker (figure 1) [39]. CD127 is another

marker negatively correlated to Treg, as it has been reported that CD127 is

down-regulated in all activated T-cell, and FOXP3+ CD127- cells account for a

high percentage of CD4+ cells in peripheral blood (figure 1) [31]. Further is this

down-regulation probably driven by FOXP3, and CD127lo/- cells which suppress

~ 17~

alloantigen in vitro [31]. T-regulatory cells 1 (TR1) are a cell population

characterized by large secretion of IL-10, but also some TGF-β, capable of

inhibiting antigen-specific immune responses and down-regulating pathological

immune response actively in mice (figure 1) [40]. Yet another cell population

considered to have regulatory properties is Th3 cells, identified in studies of oral

tolerance, producing TGF-β in large amount, however only low to none IL-4 and

IL-10, and it can induce FOXP3 (figure 1) [41-43].

Figure 1: A schematic picture of subpopulations of T-cells and the relation to different cytokines

and chemokines. Th-cells are stimulated by IL-2, IL-12, TGF-β and IFN-γ, and secrete IL-2, TNF-β.

The chemokines CXCL3, CXCL4, CXCL5, CCL9, CCL10 and CCL11 are all associated to Th1-cells.

Th2-cells are stimulated by IL-2 and IL-4, and secrete IL-2, IL-5, IL-9 and IL-13. The chemokines

CCL1, CCL2, CCL3, CCL5, CCL7, CCL11 and CCL17 are all associated to Th2-cells. nTreg-cells

express CD4, CD25, FOXP3, CD39, CTLA-4, but not CD127 and CD45RA. TR1-cells secrete IL-10,

and to some extent TGF-β. Th3-cells secrete TGF- β. Th=T-helper, nTreg=Natural regulatory T-

cell, TR1=T-regulatory cells 1, IL=Interleukin, TNF=Tumor necrosis factor, TGF=tumor growth

factor, IFN=Interferon, FOXP3=Forkhead box protein 3, CTLA-4=Cytotoxic T-lymphocyte

antigen-4.

Th1

IL-2, IL-12, TGF- , IFN-γCD4+, CD25+FOXP3+, CD39+, CTLA-4+CD127-, CD45RA-

IL-2, IL-4

IL-2, TNF-β, IFN-γCXCL3, CXCL4, CXCL5,CCL9, CCL10, CCL11

Th2

TR1 Th3

IL-2, IL-5, IL-9, IL-13CCL1, CCL2, CCL3, CCL5,CCL7, CCL11, CCL17 IL-10, TGF-β

nTreg

TGF-β

~ 18~

Cytokines and chemokines

As previously described in the “T-helper cells” section cytokines and

chemokines can be classified according to which cell type they usually are

secreted by. Besides this classification is it possible to distinguish cytokines and

chemokines with regard to their function, e.g. pro- or anti-inflammatory.

Among the pro-inflammatory cytokines are IL-1, IL-6, IL-15 and TNF-α involved

in the acute inflammation, whereas IL-1 receptor antagonist (IL-1ra), IL-4, IL-10

and IL-13 have anti-inflammatory properties [44-46]. Further, as described in

the “Regulatory T-cells” section some cytokines exert a regulatory effect. The

cytokine TGF-β is secreted not only by Th3 cells but also by FOXP3+ Treg, and

has additionally both pro- and anti-inflammatory effects, and IL-10 is secreted

by TR1 cells as described elsewhere [40, 45, 47].

~ 19~

TYPE 1 DIABETES

Pathogenesis and epidemiology

Type 1 diabetes (T1D), formerly known as diabetes mellitus, is one of the oldest

described diseases. An Egyptian manuscript written around 1550 B.C. uses the

phrase “the passing of too much urine”, and around 500 B.C. the disease was

described by an Indian physician when he noted sweetness in the urine in

certain cases. In the first century A.D. a Greek physician described the first

complete clinical symptoms, with extreme urine volumes passing through, and

he used the word “diabetes” to describe it, which is Greek and means siphon. In

1680’s an English physician adds the word “mellitus”, which is the Latin word for

honey, because of the sweetness of urine. During centuries different physicians

found more and more clues, and in 1889 Von Mering and Minkowski reported

that removal of pancreas in dogs made them develop signs and symptoms of

diabetes mellitus, and the dogs died shortly thereafter. In 1921 Banting and Best

repeated the work of Von Mering and Minkowski, and also went further when

they showed that they could reverse induced diabetes in dogs by administrating

extract from pancreatic islets of Langerhans of healthy dogs. Banting and Best

managed to purify bovine insulin together with the chemist Collip and treated

their first patient with it in 1922.

Type 1 diabetes is a chronic autoimmune disease caused by the destruction of

the insulin-producing β-cells in pancreas, leading to insulin deficiency [48]. The

lack of insulin leads to increased urination, thirst and hunger, as well as fatigue

and weight loss [49]. There is a strong genetic component in T1D, and one of the

strongest genetic associations is with human leukocyte antigen (HLA) class II, in

~ 20~

particular HLA-DQ and HLA-DR [48]. Other proposed risk genes are the insulin

gene, the protein tyrosine phosphatase, non-receptor type 22 (PTPN22) gene,

the IL-2 receptor alpha (IL-2RA) gene and the CTLA-4 gene (reviewed in [50]).

Environmental factors and viruses have also been suggested to underlie the

pathogenesis behind T1D, among them are cow´s milk, vitamin D, gluten,

psychological stress and viral infections, especially enteroviruses [50]. Still, no

single mechanism has been found to explain the pathogenesis behind T1D.

Sweden has one of the highest incidences of T1D in the world, however recent

reports show that the increase is not so steep any longer [51, 52].

Immunology

Insulitis, i.e. the destruction of the insulin-producing β-cells in the islet of

Langerhans in pancreas has been proposed to have three stages, even if the

initial event still remains unknown [53]. The first stage includes a primary

sensitization against β-cell antigens, followed by antigen-presenting cells

processing the autoantigen perpetuating the immune reaction, which causes an

inflammation in the islet. In the last stage a cycle of harmful interactions takes

place between immune- and β-cells. At least four different autoantigens have

been implicated to be important in T1D; tyrosinephosphatase (IA-2), glutamic

acid decarboxylase (GAD)65, insulin and zinc transport 8 (ZnT8) [54-57].

In the experimental murine model of T1D, non-obese diabetic (NOD) mice T1D

has been shown to be transferable by Th1-like cells [58] and in pancreas of T1D-

patients it has been shown that lymphocytes dominate, preferably IFN-γ-

secreting lymphocytes [59], implicating that T1D has a Th1-deviation. It has also

been reported that high-risk first degree relatives have a Th1-like profile up to

~ 21~

several years before disease onset, however this Th1-profile often is reduced near

or at the onset of the disease but then increases again after onset [60-63].

Children newly diagnosed with T1D have further been shown to have an

increased Th3-response, as well as an increased secretion of pro-inflammatory

cytokines [64, 65]. Pro-inflammatory and anti-apoptotic IL-15 have been

reported to be increased in serum from patients with T1D [66], and NK-cell

depleted NOD-mice treated with recombinant IL-15 delayed the disease process

and increased the suppressive functions in CD4+ CD25+ Tregs [67].

The role of Treg cells in T1D is contradictory. In 2005 Lindley et. al. reported

[68] that CD4+ CD25+ T-cells had dysfunctional suppressive properties in

comparison to healthy controls, even though frequency of them did not differ.

Further, patients with T1D had an increased secretion of IFN-γ and decreased

IL-10-secretion [68]. Brusko et. al. [69] found that the frequency of CD4+ CD25+

FOXP3+ CD127- did not differ between individuals with T1D, their first degree

relatives and healthy subjects, while insulin increased messenger RNA (mRNA)-

expression as well as frequency of CD4+ CD25+ FOXP3+ cells in children with

T1D [70]. High-risk relatives of T1D patients show decreased frequency of CD4+

CD25+ CD127- compared to healthy controls, a trend also seen in the CD4+ CD25+

FOXP3+ population [71]. This phenomena was further investigated by Badami et.

al. [72] reporting a decreased frequency of CD4+ CD25+ FOXP3+ CD127- in

subjects with T1D in comparison to individuals with celiac disease or healthy.

However, the group of T1D-patients in Badami’s study was not homogenous as

50% of the subjects in that group also had celiac disease.

~ 22~

CELIAC DISEASE


During the second century A.D. celiac disease was described for the first time.

However, the first papers regarding celiac disease was published 1888 by Samuel

Gee who described the clinical features in malabsorption in infants. During the

World War II cereals were found to be the suspected agent in celiac disease,

when Willem Karel Dicke found that the death rate due to this malabsorption

decreased to almost none after shortage of bread, but increased when bread was

available again. Between 1985 and 1987 the incidence of celiac disease increased

four times in Sweden and remained high until 1995 when it dropped sharply [73]

(figure 1). This was partly explained by changes in the infants’ dietary advices

regarding breast feeding, when to introduce gluten as well as amount of gluten

at introduction. The incidence of celiac disease still increases in Sweden and the

annual incidence was 44 cases per 100 000 person-years in 2003 [74] (figure 2).

~ 23~

Figure 2: Annual incidence rates of CD in children from 1973 to 2003. Calculations were based on

retrospective data covering 15% of the Swedish childhood population during the period from 1973

to 1990, on prospective data covering 40% from 1991 to 1997, and on data with nationwide

coverage from 1998 to 2003. Arrows indicates the start and stop of the “epidemic” of celiac

disease in Sweden. Picture adopted from Olsson et. al. 2008 [74].

Celiac disease is caused both by environmental factors as well as genetic factors.

The strongest genetic association is with HLA-genes, in particular HLA-DQ2

and HLA-DQ8 [75, 76]. In monozygotic twins the concordance has been

reported to be almost 85% in comparison to dizygotic twins where the

concordance is found to be 17% [77]. However, around 20% of the general

population in Europe carries the DQ2 heterodimer, around 50% if DQ8 is

included, and as the genetic effect of HLA is estimated to be between ~30 and

50%, other genes than HLA- DQ2-8 are involved as well [78, 79]. Among them

are genes for IL-12A, CCR1, CCR2, CCR3, CCR5 and CTLA-4 (reviewed in [80]).

The initial event leading to the development of celiac disease is still hidden, but

infections in the intestine causing a transient change in gut permeability might

be one explanation, because both bacteria and virus have been implicated in the

~ 24~

disease process. Rod-shaped bacteria have been identified in intestine of

patients with celiac disease, both untreated and treated in comparison to

controls [81]. A longitudinal study has revealed that high frequency of rotavirus

infections might increase the risk of celiac disease, which could be explained by

the similarities between VP-7, a rotavirus-neutralising protein and tissue

transglutaminase (tTG), reviewed in next section [82, 83]. Classical symptoms in

celiac disease are diarrhea, flatulence, weight loss and fatigue, however

symptoms with no clear gastrointestinal connection are also present, like

osteopenic bone disease, and so far gluten-free diet (GFD) is the only way to

avoid symptoms [84].

Immunology

Gluten is a protein with two fractions in wheat, called gliadins and glutenins,

similar proteins in barley are called hordeins and in rye secalins [85]. The

enzyme tTG has been reported to modify the gliadin peptides via deamidation,

thereby binding gliadin with higher affinity to HLA-DQ2 and HLA-DQ8,

causing an epitope that is recognized by T-cells derived from the gut and

increasing gliadin-specific reactivity of T-cells [86, 87]. One explanation for the

immunological process is that native gluten bound HLA-DQ2 or HLA-DQ8 is

presented to CD4+ T-cells via antigen presenting cells leading to secretion of

IFN-γ, which in turn leads to increased expression of HLA-DQ molecules. If this

loop leads to tissue damage and subsequent release of tTG, thereby modifying

the affinity of gliadin, and increasing the CD4+ T-cell population, then this leads

to more tissue damage and initializes a secondary loop (figure 3) [80]. One

alternative explanation is that infections in the intestines generate a pro-

inflammatory response with a resulting loss of tolerance to gluten, generating

additional tissue damage and initiating deamidation [80]. The infiltration of

~ 25~

inflammatory cells leads to lesions in the intestine of patients with celiac

disease, characterized by villous atrophy and crypt hyperplasia [85].

Figure 3: A model of a self-amplifying loop leading to development of celiac disease. Presentation

of native peptides of gluten increases IFN-γ secretion from CD4+ cells, with an increase of HLA-

DQ2 and HLA-DQ8 expression as result. This might lead to tissue damage with release of tTG,

leading to a secondary loop with further increase of IFN-γ secretion and HLA-DQ2 and HLA-

DQ8. IFN=Interferon, HLA=Human leukocyte antigen, tTG=Tissue transglutaminase. Picture

adopted from Tjon et al. 2010 [80].

Usually three autoantibodies are seen in patients with celiac disease, antibody

against gliadin (AGA), endomysium (EMA) and tissue translutaminase (tTGA)

[88-90]. They differ in sensitivity and specificity, and also with age, and are used

in combinations to diagnose celiac disease [91, 92]. Celiac disease is believed to

have a Th1-deviated immune response, the intestinal mRNA-expression of Th1-

associated cytokines IL-2, IFN-γ and TNF-β as well as the secretion of those

cytokines in peripheral blood has been observed to be increased [93].

Addtionally, mRNA levels of IFN-γ and IL-10, but not IL-4 have been reported to

be increased in intestine from patients with untreated celiac disease [94, 95]. In

a recent study with aim to study what effect GFD has on Th1-, Th2- and Th3-

CD4 T cell IFN-γ

HLA-DQ2/8 Deamidated gluten peptidesGluten presentation

HLA-DQ2/8

CD4 T cell

Possible tissue damage

tTG

~ 26~

associated response in small intestine as well as in peripheral blood it was

reported that patients with an untreated celiac disease have an increased Th1-

response, but a decreased Th2-response [96]. After one year on GFD the Th2-

response was normalized, but the alterations seen in mucosa were not reflected

in peripheral blood. There is an increase of the pro-inflammatory cytokine IL-15

in patients with an untreated celiac disease in comparison to patients with

treated disease or controls, which has been reported repeatedly [97-99]. Also

FOXP3 has been reported repeatedly [100-102] to be increased in patients with

an untreated celiac disease, both at the level of mRNA-expression and frequency

of CD4+ CD25+ FOXP3+ cells.

~ 27~

ALLERGY


Already during antiquity allergic diseases were described, but the term allergy

was introduced first in 1906 by Clemens von Pirquet from the two ancient Greek

word “allos” meaning other and “ergon” meaning work. In 1916 Cooke and van

der Veer described skin reactions, and during the 1960’s the different

hypersensitivity reactions were described for the first time by Gell and Combs.

Only type I hypersensitivity was now considered allergic, and in 1967 the

antibody IgE was described for the first time by Ishizaka and Johansson. The

term “atopy” was used by Coca and Cooke for the first time 1923, from Greek

meaning out of place, special and unusual, and atopy was for a long time the

term for any IgE-mediated reaction. The term atopy has further been classified

to describe the hereditary predisposition to become sensitized and produce IgE

antibodies in response to allergens, while allergy is classified as a

hypersensitivity reaction initiated by immunological mechanisms [103]. Typical

allergic symptoms include asthma, rhinitis, conjunctivitis, dermatitis (both

eczema and contact dermatitis), urticaria and gastrointestinal symptoms. When

the symptoms are IgE-driven they are called atopic followed by appropriate

symptom [103]. The term “atopic march” is used for describing the variation of

allergic symptoms with age. It starts with atopic dermatitis and food allergy

during the first 2 years of life, followed by vanishing of food allergies and

progression to asthma and rhinocunjunctivis in pre-school age [104, 105].

Approximately 50% of the children with atopic dermatitis will develop asthma

later on [104, 105].

~ 28~

During the years 1979 to 1991 the prevalence of asthma, allergic rhinitis and

eczema was doubled among Swedish schoolchildren [106]. Environmental

factors are believed to have a high impact on the development of allergy and

asthma, as reported by several groups, among them the International Study of

Asthma and Allergies in Childhood (ISAAC) as well as comparisons between

Sweden and Estonia. Atopic sensitization and asthma symptoms examined in

the ISAAC material have been shown to increase with economic development

[107]. Also by a direct comparison of atopy and allergic disorders in children and

adults in Sweden and Estonia it was found that the prevalence of atopy was

increased in Sweden proposed to be caused by low endotoxin levels [108]. This

indicates that a reduced pressure to different microbes increases allergic disease

[109]. Additionally, exposure to tobacco smoke, animals, farming, lactobacilli,

fatty acids, day care and antibiotic use have been proposed to be involved in

allergic diseases [110-115].

Immunology

The IgE-mediated immune response is commonly divided into sensitization,

early or immediate hypersensitivity reaction and late phase reaction [116, 117]. In

the sensitization step specific IgE antibodies are synthesized by plasma cells,

with involvement of allergen-specific Th2-cells. Produced IgE-antibodies are

mainly found on mast cells. The immediate hypersensitivity reaction takes place

within minutes from the re-exposure to the allergen. Then the allergen binds

and cross-links to IgE-sensitized mast cells, which results in a release of

inflammatory mediators like histamine, tryptase and cytokines, triggering

different responses including vascular permeability, broncho-constriction and

production of mucus. The different cytokines and chemokines, for example IL-4,

~ 29~

IL-5, IL-9 and IL-13 drive the late allergic response where eosinophils, basofils

and Th2-cells are recruited to the inflammation site.

Allergic disease and asthma are considered being Th2-deviated, as shown by

atopic asthmatics having an increased mRNA-level of IL-3, IL-4, IL-5 and

granulocyte-macrophage colony-stimulating factor (GM-CSF) in

bronchoalveolar lavage (BAL), in comparison to controls, and IL-4 and IL-13

induce the switch to IgE [118], There is also decreased frequency of IFN-γ-

producing cells in CD4+ cells, but not CD8+ cells, in atopic individuals [119].

Additionally, the Th1-associated cytokine IL-12 has the ability to suppress

synthesis of IgE in IL-4-stimulated peripheral blood mononuclear cells (PBMC)

[120], and it also dampens the airway hyperresponsiveness in mice [121]. The TR1-

associated cytokine IL-10 has been shown to be increased in monocytes in atopic

patients [122], and has also been reported to be increased during specific

immune therapy (SIT) [123].

~ 30~

COMBINATIONS OF TYPE 1 DIABETES, CELIAC DISEASE AND ALLERGY

According to the theory that Th1 and Th2 are in balance with each other, a Th1-

deviated disease should not co-exist with a Th2-deviated disease. However, this

is not the case as individuals might suffer from example T1D or celiac disease

and allergy at the same time, and there are also cases with all three diseases in

combination.

Combination of type 1 diabetes and celiac disease

Besides a common immunological profile, with a Th1-devation, individuals with

T1D and celiac disease share some risk genes, i.e HLA-DQ2 and HLA-DQ8

leading to increased risk of developing the other disease once affected by one of

these diseases [124]. Screening children with T1D for non-islet autoantibodies

revealed that 11,6% had tTG autoantibodies, but only just around a quarter of

them had developed celiac disease [125]. Besides a common genetically

predisposition, gliadin sensitivity has been reported in both T1D and celiac

disease [86, 126, 127]. There are studies showing that the gut is involved in T1D,

both with regard to a change in permeability and with regard to mucosal

immunity. Children with T1D and no sign of celiac disease have been reported

to have both increased expression of HLA class II genes, and an increase of

adhesion molecule in mucosa, and additionally an expression of gut-associated

homing receptor α4β7-integrin in GAD-specific T-cells suggesting an interplay

between gut and pancreas [128, 129]. This has been further supported by other

groups, suggesting that gluten, or gliadin, has the property of increasing the

permeability of the gut both in mice and humans [126, 127, 130, 131]. Further does

the FOXP3 mRNA-expression, as well as the frequency of FOXP3+ cells in small-

~ 31~

bowel mucosa, increase when celiac disease is accompanied with T1D in

comparison to individuals with T1D or celiac disease as single diseases [132].

Combination of type 1 diabetes and allergy

Most studies on T1D and allergy focus on the negatively inverse relationship

between those diseases, with regard to T1D being a Th1-associated disease and

allergy being a Th2-associated one [133]. A study by Gazit et. al. reported that

the prevalence of atopy in T1D-patients was similar to the general population

[134], while Olesen et. al. reported that atopic dermatitis was decreased in

patients with T1D if atopic dermatitis was developed first [135]. Further, children

with combination of T1D and asthma have been shown to have a similar pattern

of IFN-γ, IL-2, IL-4, IL-10 and IL-13 in PBMC after stimulation with

phytohaemagglutinin (PHA), tetanus toxoid (TT) or anti-CD3 monoclonal

antibodies [136]. The IL-10 secretion in total (both spontaneous and stimulated

secretion) was increased in the group with combination of T1D and asthma

[136]. Kainonen et. al. [137] reported that spontaneous IFN-γ, TNF-α and IL-10

were higher in children with both T1D and asthma, however stimulation with

CD3 and CD28 did not increase the secretion of IL-10 in this group. Finally

serum levels of IL-12 and IL-18 have been shown to be increased in children with

T1D and asthma, as well as the serum ratio of IL-18/IL-12, while stimulation with

lipopolysaccharide (LPS) decreased the IL-12 secretion [138].

Children with combination of celiac disease and allergy

Children with celiac disease and allergy have the same difference in

immunological profile as patients with T1D and allergy, with celiac disease

~ 32~

having a Th1-associated profile and allergy a Th2-associated profile. In a Finnish

study from 1983 Verkasalo et. al. [139] found that children with celiac disease

had an increase of atopy in comparison to unselected schoolchildren, and atopy

was more frequently seen in individuals carrying HLA-B8DR3-. In Italy the

prevalence of silent celiac disease in atopics has been reported to be 1%, which is

significantly higher than the general Italian population [140]. The cumulative

incidence of asthma is significantly higher in children with celiac disease and

allergy in Finnish children compared to children with exclusively celiac disease

[141]. Recently a study performed in Sweden reported that there is a 1.6-fold

increased risk of developing asthma when you suffer from celiac disease, as seen

by the hazard ratio [142].

~ 33~

CRYOPRESERVATION

Cryopreservation is a convenient way to collect and store samples, e.g. PBMC or

tissue. Through this process it is possible to collect the samples during a limited

period of time, and by performing all analyses collectively it is possible to over-

come inter-assy variation. However, the freezing and thawing of cells is a harsh

process and might harm or change the sample. Studies performed on the

cytokine IL-10 have shown contradictory results, with either a decrease or no

change at all after cryopreservation [143-145]. The same pattern has been

reported for frequency for the Treg-associated marker FOXP3, ranging from a

significant decrease to no change at all [146-148]. Both freezing protocols and

used medium can affect the cryopreservation, which might be an explanation

for differences in result [147, 149].

~ 35~

AIMS OF THE THESIS

The general aim of this thesis was to study the immune profile with focus on subsets of T-lymphocytes in children with T1D, celiac disease, allergy, a combination of two of these diseases and healthy children (reference).

The specific aims of the papers were:

Paper I: To evaluate the secretion of the Th1-associated cytokine IFN-γ and the Th2-associated cytokine IL-4 in PBMC, both spontaneously and after in vitro stimulation with disease-associated antigens, in children with T1D, celiac disease, allergy, combination of two of these as well as reference children.

Paper II: To evaluate the secretion of cytokines and chemokines in cell supernatant, both spontaneously and after in vitro stimulation with disease associated antigens, in children with T1D, celiac disease, allergy, two of these diseases in combination in comparison to reference children.

Paper III: A methodological study to examine the mRNA-expression of markers associated with regulatory T-cells (i.e. FOXP3, TGF-β, CTLA-4 and sCTLA-4), and additionally comparison between 48- and 96-hours stimulation period, both spontaneous and after antigen-stimulation.

Paper IV: To examine the mRNA-expression of the Treg-associated marker FOXP3 after in vitro stimulation in children with T1D, celiac disease, combination of T1D and celiac disease and reference children.

Paper V: To evaluate the expression of markers associated with regulatory T-cells (CD4, CD25, CD39, FOXP3, CD45RA and CD127) in children with T1D, celiac disease, combination of T1D and celiac disease and reference children.

~ 37~

MATERIAL AND METHODS

Study population

The study population in this thesis (paper I-V) consists of three different

cohorts, see figure 4. In paper I, II and IV the same cohort was included with

slightly different compositions, with children from Linköping University

Hospital, Sweden. In paper II the cohort included healthy adults from

Linköping, Sweden. Paper V included children from Ryhov Hospital, Jönköping,

Sweden.

Cohort 1, paper I and II describes children diagnosed with T1D, celiac disease,

allergy or a combination of two of these diseases, in relation to healthy children

as reference. Cohort 1, paper IV describes children diagnosed with T1D, celiac

disease or a combination of these diseases, in relation to healthy children as

reference. Cohort 2, paper III describes healthy adults from Linköping, Sweden.

Cohort 3, paper V describes children diagnosed with T1D, celiac disease or a

combination of these diseases, in relation to healthy children as reference.

Figure 4: A description of cohorts used in the different papers.

Cohort 1 Cohort 2

Paper II

Cohort 3

Paper I Paper IIIPaper IV Paper V

~ 38~

Paper 1

Sixty-eight children from cohort 1 were included in the study, diagnosed with

either T1D, celiac disease or allergy, or a combination of two of these diseases.

Healthy children, i.e. without any of these diseases were used as reference

children. All children were matched by gender and age as closely as possible, see

table I. This material includes all children diagnosed with both T1D and celiac

disease, at the Paediatric clinic at the University hospital of Linköping, Sweden,

i.e. 4.4% (8/180). These children represent the Swedish population since 4.6 %.

of the patients with celiac disease in Sweden have been found to have T1D, at

the time for inclusion.

Table 1 Children with type 1 diabetes, celiac disease and/or allergy and reference children

Gender T1D+CD T1D+AD CD+AD T1D CD AD Ref

Boys 14 (3 y/ 4 y) - 11 (1 m) 13 (4 y) - 13 12

13 (18 m/ 5 m) 12 (10 m) - 12 (11 y) 10 (d.n.a.) 12 12

12 (8 y/ 18 m) 12 (8 y) - 12 (2 y) 10 (1 y) 9 10

10 (3 y/ 9 m) 9 (4 y) 10 (6 y) 9 (5 y) 8 (6 y) 9 9

7 (1 y/ 9 m) 6 (4 m) - 6 (1 y) 7 (4 y) 6 8

- - - 13 (3 m) - - -

Girls - 14 (11 y) 16 (4 m) 14 (3 y) 15 (11 y) 14 14

12 (2 y/ 1 y) 12 (5 y) 14 (12 y) 12 (7 y) 14 (11 y) 12 13

- 12 (5 y) 11 (9 y) 12 (10 y) 12 (1 d) 12 12

10 (5 y/ 10 m) 10 (6 y) 11 (8 y) 10 (14 m) 11 (9 y) 11 12

8 (2 y/ 10 m) 10 (7 y) 10 (6 y) 10 (2 y) 10 (9 y) 11 11

- - - 10 (9 m) 10 (7 y) 10 -

- - - - - 12 -

Range (mean) 7-14 (10.8) 6-14 (10.8) 10-16 (11.9) 6-14 (11.1) 8-15 (10.7) 6-14 (10.9) 8-14 (11.3)

No 8 9 7 12 10 12 10

Children with type 1 diabetes (T1D), celiac disease (CD) or allergy (AD), and combinations of these diseases (T1D+CD, T1D+AD, CD+AD)

were matched to each other and to reference (REF) children by age (years) and gender. Duration of disease (in parenthesis d = days, m = months,

y = years) is presented for children with T1D and/or CD.

d.n.a.= data not available

~ 39~

Paper II

The study group in paper II consisted of 72 children from cohort 1, either

diagnosed with T1D, celiac disease, allergy or a combination of two of these

diseases. Children without any of these diseases were used as reference group,

see table II. All children were matched by gender and age as closely as possible.

Paper III

Twelve healthy adults (median age 31 years of age, 25-52 years, 7 female/5 male)

from cohort 2 comprised the study group for paper III.

Table II Children with type 1 diabetes, celiac disease and/or allergy and reference children

Gender T1D CD AD T1D + CD T1D +AD CD + AD Ref

Boys 12 (11 y) 10 (d.n.a.) 12 13 (18 m/5 m) 12 (10 m) 12

12 (2 y) 10 (1 y) 9 12 (8 y) 10

9 (5 y) 8 (6 y) 9 10 (3 y/9 m) 9 (4 y) 10 (6 y) 9

6 (1 y) 7 (4 y) 6 7 (1 y/9 m) 6 (4 m) 8

13 (3 m) 15 (d.n.a.) 12

13 14 (3y/4 y) 11 (1 m) 12

12

14

Girls 14 (3 y) 15 (11 y) 14 14 (11 y) 16 (4 m) 18

12 (7 y) 14 (11 y) 12 12 (2 y/1 y) 12 (5 y) 14 (12 y) 18

12 (10 y) 12 (1 d) 12 11 (d.n.a.) 12 (5 y) 11 (9 y) 12

10 (14 m) 11 (9 y) 11 10 (5 y/10 m) 10 (6 y) 11 (8 y) 10

10 (2 y) 10 (9 y) 11 8 (2 y/10 m) 10 (7 y) 10 (6 y)

10 (9 m) 10 (7 y) 10 13

11 (d.n.a.) 12

Range (mean)6-14 (10.9) 7-15 (10.7) 6-14 (10.9) 7-15 (11.1) 6-14 (10.8) 10-16 (11.9) 8-18 (12.3)

No 12 10 12 9 9 7 13

Children with type 1 diabetes (T1D), celiac disease (CD) or allergy (AD), and combinations of these diseases (T1D+CD, T1D+AD, CD+AD)

were matched to each other and to reference (REF) children by age (years) and gender. Duration of disease (in parenthesis d = days, m = months,

y = years) is presented for children with T1D and/or CD.


~ 40~

Paper IV

Paper IV included 24 children from cohort 1, diagnosed with T1D, celiac disease,

T1D and celiac disease in combination as well as reference children. All children

were matched by gender and age as close as possible. See table III.

Table III: Children with type 1 diabetes, celiac disease and/or allergy and reference

children

Paper V

Thirty-two children from cohort 3, diagnosed with T1D and/or celiac disease

were included in this study. Children without any of these diseases were used as

reference (table IV). The material includes children with T1D and celiac disease

Gender T1D+CD T1D CD REF

Boys 14 (3 y/ 4 y) 12 (2 y) 10 (d.n.a.) 12

10 (3 y/ 9 m) 10 (1 y) 12

7 (1 y/ 9 m) 8 (6 y) 9

7 (4 y) 8

Girls 12 (2 y/ 1 y) 12 (7 y) 15 (11 y)

10 (5 y/ 10 m) 12 (10 y) 14 (11 y)

10 (14 m) 12 (1 d)

10 (2 y) 11 (9 y)

10 (9 m) 10 (9 y)

Range (mean) 7-14 (10.6) 6-14 (11) 7-15 (10.8) 8-18 (10.3)

N 5 6 9 4

Children with type 1 diabetes (T1D), celiac disease (CD) and combination of T1D and CD

were matched to each other and to reference (REF) children by age (years) and gender.

Duration of disease (in parenthesis d = days, m = months, y = years) is presented were available.


~ 41~

from the pediatric clinic, Ryhov Hospital, Jönköping, Sweden. The combined

T1D and celiac disease group was the first to be included, and as far as possible

the other groups were matched by age and gender to this group.

Table IIII: Children with type 1 diabetes, celiac disease and/or allergy and reference

children

Children with type 1 diabetes (T1D), celiac disease (CD) and combinations of these diseases (T1D+CD)

were matched to each other and to healthy children by age (Y=years) and gender.

Diagnostic criteria

Celiac disease was diagnosed according to the modified version of The European

Society of Paediatric Gastroenterology, hepatology and Nutrition (ESPGHAN),

former The European Society of Paediatric Gastroenterology and Nutrition

(ESPGAN) criteria [150]. Duration of T1D was defined as the date of diagnosis

and celiac disease as the date of the first biopsy. For diagnosis of allergy we

strictly adhered to the protocols for phase I and III described in the ISAAC study

(http://isaac.auckland.ac.nz/phases/phaseone/phaseone.html, and

http://isaac.auckland.ac.nz/phases/phasethree/phasethree.html). Allergy was

T1D + CD T1D CD Ref

male 8 7 8 7

female 8 8 - 8

female 8 13 10 10

male 11 15 10 14

female 13 14 13 10

female - 14 - 11

male 14 15 - 14

female - 14 17 14

male 16 17 - -

female - 17 17 16

No n=7 n=10 n=6 n=9Range (mean) 8-16 y (11.1) 7-17 y (13.4) 8-17 y (12.5) 7-16 y (11.6)

~ 42~

characterized by eczema, bronchial asthma and/or allergic rhinoconjunctivitis.

Skin prick test (SPT) was performed on all children in duplicate on the volar

aspects of the forearms with standardised cat and dog dander extracts, birch,

timothy, grass, mite (Soluprick R, ALK, Hørsholm, Denmark) and hen’s egg

white. Tests were regarded as positive if skin wheals had a mean diameter of 3

mm or more after 15 minutes. Histamine dihydrochloride, 10 mg/ml, and a

sterile lancet were used as positive and negative controls respectively. The

reference children had no signs of T1D or of any other autoimmune disease, nor

any clinical signs of celiac disease or clinical allergy, and the SPT was negative

for all of them. Further, no signs of these diseases were reported among their

first-degree relatives. None of the children had signs of a cold or other infection

at the time of blood sampling.

Laboratory analyses

Isolation and in vitro stimulation of PBMC

In paper I, II, III and IV PBMC were isolated from sodium-heparinised venous

blood samples with Ficoll Paque (Pharmacia, Biotech, Sollentuna, Sweden),

within four hours from blood sampling. After density gradient centrifugation for

30 minutes at 400 g, mononuclear cells were collected at the interface between

blood and Ficoll, followed by wash two times by centrifugation for 15 minutes at

400 g in RPMI 1640 medium (Gibco, Täby, Sweden), supplemented with 2% fetal

calf serum (FCS) (Gibco). Cells were stained with Türks solution (1:10) and

counted in Bürkers chamber by light microscopy, followed by a final wash. For

cells intended to be cultured, PBMC were diluted to 2 x 106 PBMC/ml in AIM V

serum free medium supplemented with 2 mM L-glutamine, 50 μg/ml

streptomycin sulphate, 10 μg/ml gentamycin Sulphate and 2 x 10-5 M 2-

mercaptoethanol (Gibco, Täby, Sweden) and cultured as follows:

~ 43~

In paper II PBMC were cultured in vitro for 96 hours with insulin 500 µg/ml

(Actrapid, Novo Nordisk, Denmark), gluten 400 µg/ml (gift from Outi Vaarala,

Helsinki, Finland) and birch 10 kSU/ml (Aquagen, ALK-Abell, Hærsholm,

Denmark). Medium alone was used for detection of spontaneous secretion, and

PHA 5 µg/ml (Sigma, Stockholm, Sweden) was used as positive control. Cell

supernatants were collected and frozen at -70°C until further analysis.

In paper IV PBMC were cultured in vitro for 96 hours with tTG 500 μg/ml,

gluten 400 μg/ml (both gifts from Outi Vaarala, Helsinki, Finland) and insulin

500 μg/ml (Actrapid, Novo Nordisk, Denmark). Phytohaemagglutinin 5 μg/ml

[61] (Sigma-Aldrich, Stockholm, Sweden) was used as a positive control and

medium alone for detection of spontaneous mRNA expression. In samples with

limited number of cells, the order of priority for stimulation with antigens was

PHA, gluten, tTG and finally insulin. Cells were collected post-stimulation and

stored at -70°C for further analysis of mRNA expression.

Freezing, thawing and in vitro stimulation of PBMC (paper III)

After counting PBMC were centrifuged at 400 g for 10 minutes and freezing

medium containing 50% RPMI 1640 without glutamine (Gibco, Invitrogen

Corporation, UK), 40% FCS (Gibco, Invitrogen Corporation) and 10% dimethyl

sulfoxide (DMSO) (Sigma) was added while tubes were agitated. Cells with a

concentration of 5-10 x 106 PBMC/ml were moved to cryo vials and placed in a

pre-cooled (-4° C) Cryo 1°C Freezing Container (Nalge Nunc International,

Rochester, USA) containing isopropanol. The container was placed in -70°C with

a freezing rate of -1°C/minutes. The following day the vials were transferred to

liquid nitrogen (-196°C) and stored frozen until analysis.

~ 44~

After storage in liquid nitrogen for at least one month, PBMC were quickly

thawed to 37°C in water bath, and cell suspensions were immediately washed

with RPMI supplemented with 10% FCS. The cell suspensions were centrifuged

for 10 minutes at 400 g, then the suspensions were removed and the cell pellets

were resuspended in AIM-V serum free medium (Gibco, Täby, Sweden)

supplemented with 2 mM L-glutamine, 50 μg/ml streptomycin sulphate, 10

μg/ml gentamycin Sulphate and 2 x 10-5 M 2-mercaptoethanol (Sigma-Aldrich,

Stockholm, Sweden). Peripheral blood mononuclear cells stained with Türks

solution were counted in phase contrast microscopy and viability was calculated

with trypan blue. A viability level of at least 90% was considered acceptable.

Stimulation of frozen and thawed PBMC was performed following the same

stimulation protocol as for freshly stimulated cells, and the same procedure was

followed for collection of PBMC and cell supernatant.

Cell concentration was adjusted to 2 x 106 PBMC/ml with AIM-V serum free

medium (Gibco, Sweden) supplemented with 2 mM L-glutamine, 50 μg/ml

streptomycin sulphate, 10 μg/ml gentamycin Sulphate and 2 x 10-5 M 2-

mercaptoethanol (Sigma-Aldrich, Stockholm, Sweden), and PBMC were

stimulated with PHA (Sigma-Aldrich), TT (SBL, Stockholm, Sweden), β-

lactoglobulin (βLG) (Sigma-Aldrich) and ovalbumin (OVA) (Sigma-Aldrich) for

48 and 96 hours respectively at 37°C, in 5% CO2 (for concentration see table V).

Antigen Concentration for 48 h Concentration for 96 h

PHA 5 µg/ml 5 µg/ml

TT 20 Lf/ml 20 Lf/ml

βLG 5 µg/ml 50 µg/ml

OVA 5 µg/ml 50 µg/ml

Table V Concentration of antigens.

Concentration for 48 and 96 h stimulation period.

PHA=Phytohaemagglutinin. TT=Tetanus toxoid.

βLG=Betalactoglobulin. OVA=Ovalbumin.

~ 45~

Collection of PBMC and cell supernatants (paper II, III and IV)

After centrifugation for 10 minutes at 400 g aliquots of cell supernatants were

removed and PBMC were resuspended in AIM-V serum free medium (Gibco,

Sweden) supplemented with 2 mM L-glutamine, 50 μg/ml streptomycin

sulphate, 10 μg/ml gentamycin Sulphate and 2 x 10-5 M 2-mercaptoethanol

(Sigma-Aldrich, Stockholm, Sweden). Both cell suspension and cell supernatant

were kept frozen at -70°C until further analysis.

Enzyme linked immunospot (ELIspot) (paper I)

In an ELIspot assay it is possible to study cytokine secretion at the single cell

level, as the released cytokine is captured by antibodies bound to the surface of

a microtiter plate before it is digested or internalised by the cells (figure V).

Sterile 96-well microtiter plates (Multiscreen HA, Milipore, Bedford,

Massachusetts, USA) were precoated with 70% ethanol, followed by five times

wash with sterile water. Coating antibody, i.e. mouse anti-human IFN-γ 7B61

(Mabtech AB, Stockholm, Sweden) or mouse anti-human IL-4 MoAB (Mabtech

AB) was added to the wells, and the plates were incubated overnight at 4°C in a

humid chamber. After wash with phosphate buffer saline (PBS), the wells were

incubated for 30 minutes in 37°C and 5% CO2 with Iscove´s modification of

Dulbeccos medium (IMDM) (Life Technologies, Täby, Sweden) containing 50

U/ml penicillin (Life Technologies), 50 µg/ml streptomycin (Life Technologies),

10 ml/l 100 x MEM non-essential amino acids (Life Technologies) and 5% FCS

(Gibco) to block unspecific binding sites. After removal of IMDM cells with a

concentration of 1 x 105 PBMC/well were added together with 100 µl IMDM with

supplements as well as antigens according to table VI and incubated

undisturbed for 48 hours, at 37°C and 5% CO2 in a humid chamber. Border wells

of the plates contained exclusively IMDM and functioned as a negative control,

~ 46~

while PHA functioned as a positive control. All stimulated and unstimulated

samples were applied in quadruplicates, and all plates were blinded for

identification of samples.

After 48 hours incubation the plates were washed twice with sterile PBS and

twice with PBS Tween and incubated for two hours, at room temperature (RT),

in darkness and in a humid chamber, with biotinylated anti-human IFN-γ 1-

D1KmoAb (Mabtech AB) or bionibylated anti-human IL-4 MoAB (Mabtech AB)

respectively, both with a concentration of 1 µg/ml. After wash with PBS Tween

streptavidin conjugated with alkaline phosphatase (AP) (Mabtach AB), diluted

1:1000, was added to the plates, followed by incubation for one hour, in humidity

at RT. After wash twice with PBS Tween and four times with PBS spots were

developed by AP conjugate substrate kit (Bio-Rad, Hercules, California, USA)

diluted 1:25. The development process proceeded for 10-15 minutes and the

plates were washed with abundant amounts of tap water, emptied and placed

upside down in darkness to dry overnight at RT.

Table VI Antigens

Antigen ConcentrationSource

Birch extract 10 kSQ-U/ml Aquagen, ALK-Abelló, Hørsholm, Denmark

Cat allergen extract 10 kSQ-U/ml " "

Insulin 500 ng/ml Actrapid, NovoNordisk, Malmö, Sweden

β-lactoglobulin 10 mg/ml Sigma-Aldrich, Stockholm, Sweden

Ovalbumin 10 mg/ml " "

Gluten 400 mg/ml Gift from O Vaarala, Linköping, Sweden

Phytohaemagglutinin 20 mg/ml Sigma-Aldrich, Stockholm, Sweden

Concentration and source of antigens used for stimulation of peripheral blood

mononuclear cells (PBMC) to induce antigen-specific secretion of interferon(IFN)-γ

and interleukin(IL)-4, detected by enzyme linked immunspot (ELIspot)-technique.

~ 47~

Plates were counted automatically, by the AID ELISPOT Reader System (AID,

Strasbourg, Germany), where each well was photographed and spots were

marked based on size, colour and intensity. However, artefacts were sometimes

regarded as spots, hence manual supervision was required. The median value of

the quadruplicates for each antigen was calculated and the value of secretion for

unstimulated PBMC (i.e. spontaneous secretion) was subtracted from the

secretion from stimulated PBMC in order to obtain antigen-specific secretion.

Our laboratory participated in the first ELISPOT workshop as one of the core

laboratories, and our assay was judged to be sensitive and reproducible [151].

Multiplex fluorochrome technique (Luminex) (paper II)

With Luminex technique it is possible to fast and precisely analyse up to 100

simultaneous tests in one single reaction. It is a sandwich immunoassay, based

on microspheres individually dyed (figure 6). On the surface of the microsphere

antibodies directed against analyte of interest are bound. When the

microspheres pass through a laser, the combination of dye is read for

identification of analyte, while another laser reads the amount antibody bound

to each sphere. A standard curve is used to correlate each concentration to the

unknown sample.

Figure 5: Principle for the enzyme linked immuno spot (ELIspot) technique. Capture antibody, eitherinterferon (IFN)-γ or interleukin (IL)-4 were bound to the bottom wells in a microtiter plate. Cells were allowed to react to antigens, hereby secreting cytokines. Biotinylated detection antibodies (IFN- γ or IL-4)

were added. After incubation, streptavidin conjugated with alkaline phosphatase (AP), and thereafter developed by AP conjugate. Picture published after kind permission from Jenny Walldén.

~ 48~

Figure 6: Principle of the multiplex fluorochrome technique (Luminex). Antibody coupled bead,

individually dyed, binds to analyte. A biotinylated detection antibody is added and finally

streptavidin-phycoerythrin (PE) is added and samples are analysed.

In paper II Luminex technique was used to analyse the secretion of IL-1β, IL-1ra,

IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, CCL11,

fibroblast growth factor (FGF)Basic, granulocyte colony-stimulating factor (G-

CSF), GM-CSF, IFN-γ, CXCL10, CCL2, CCL3, CCL4, platelet-derived growth

factor-bb (PDGFbb), CCL5, TNF-α and vascular endothelial growth factor

(VEGF) in cell supernatants. A Bio-PlexTM Human Cytokine 27-plex panel (Bio-

Rad Laboratories, Hercules, CA, USA) and The BioPlexTM Cytokine Reagent Kit

(Bio-Rad Laboratories) were used according to the manufacturer´s instructions.

Sample, blank, standards and beads were added to 96-well microtiter plates,

samples as singleplex, blank and standard curve in duplicates, and incubated for

30 minutes at RT. After wash with a vacuum device, biotinylated detection

antibody was added, followed by 30 minutes incubation at RT and another

wash. Streptavidin-phycoerythrin (PE) was added to the plate, and after

incubation for 10 minutes at RT an additional wash was performed. After

resuspension of the beads in assay buffer, the samples were analysed in a

Luminex 100TM instrument (Luminex xMAPTM Technology, Austin, TX, USA).

The threshold for acquisition was set to a minimum of 100 beads per region.

Raw data (median fluorescence intensity, (MFI)) for each reaction were analysed

using StarStation Software v 2.3 (Applied Cytometry Systems, Sheffield, UK). To

exclude possible interference of culture medium, wells including only AIM V

Y Y

Y

Y Y

Y

~ 49~

serum free medium (Gibco, Täby, Sweden) were subtracted from each sample.

In order to obtain antigen specific response the spontaneous secretion was

subtracted from stimulated secretion. A five-parameter curve fit was applied to

each standard curve to acquire sample concentration values. For cut off values,

see table VII.

Cut-off values for cytokines and chemokines included in the Luminex-assay. FGF=Fibroblast

growth factor, G-CSF=Granulocyte colony-stimulating factor, GM-CSF= Granulocyte

macrophage colony-stimulating factor, IFN=Interferon, IL=Interleukin, PDGF=Platelet-derived

growth factor, TNF=Tumor necrosis factor, VEGF=Vascular endothelial growth factor.

Table VII

Cytokine/Chemokine Cut-off (min) (pg/ml)

CCL2 0,4

CCL3 0,3

CCL4 0,5

CCL5 0,5

CCL11 0,2

CXCL10 1,2

FGFBasic 0,3

G-CSF 0,6

GM-CSF 0,1

IFN-γ 0,3

IL-1β 0,5

IL-1ra 0,7

IL-2 0,3

IL-4 0,07

IL-5 0,6

IL-6 0,4

IL-7 0,8

IL-9 0,4

IL10 0,4

IL-12(p70) 0,6

IL-13 0,9

IL-15 0,4

IL-17 0,8

PDGFbb 0,2

TNF-α 0,8

VEGF 0,5

~ 50~

Real-time reversed-transcriptase polymerase chain reaction (RT-PCR)

(paper III and IV)

RNeasy 96 kit (Qiagen, KEBO, Spånga, Sweden) was used to isolate total RNA

from PBMC, according to manufacturer’s recommendation. Thawed PBMC were

lysed, mixed 1:1 with 70% ethanol and placed on RNAeasy 96-well plates. Several

washing steps including centrifugation with wash buffer included in the kit,

were performed and the RNA was eluted with RNAse-free water. Quantification

of RNA concentration was performed using a NanoDrop® ND-1000

spectrophometer (NanoDrop Technologies, Willington, Delaware, USA) at optic

density (OD) 260 nm. To determine purity of RNA the quota between OD 260

nm and 280 nm was calculated (≥ 1.8 in all cases). RNA was stored at -70°C until

further analysis.

Using equal amounts of total RNA (7 ng/μl), stimulated under various

conditions, mRNA was marked complementary with random hexamers, and

complementary DNA (cDNA) was synthesised from the mRNA by High Capacity

cDNA archive kit (Applied Biosystems, Stockholm, Sweden), according to

supplier’s recommendation. RNAse inhibitor (1 U/ml) (Applied Biosystems) was

added to the reaction, apart from reactants supplied in the kit. The synthesis

was performed using random hexamers and deoxyribonucleotide triphosphate

(dNTP). The reaction mixture was synthesised with a Gene Amp PCR System

2700 (Applied Biosystems), for 10 minutes at 25°C and at 37°C for 120 minutes.

The final cDNA product was stored at -20°C until cDNA amplification.

Transcription levels of markers associated with Treg cells (paper III: FOXP3,

TGF-β, CTLA-4 and sCTLA-4, paper IV: FOXP3) were detected by multiplex

real-time RT-PCR. TaqMan Universal PCR 2x Master Mix (Applied Biosystems),

specific primer and probe (Foxp3: Hs00203958, TGF-β: Hs00171257 and CTLA-4:

~ 51~

Hs00175480, all FAM dye layer, Applied Biosystems), s-CTLA-4 primers and

probe (FAM dye layer, synthesised by Applied Biosystems) and endogenous

reference primers and probe (Human 18s ribosomal RNA (rRNA): 4310893E, VIC

dye layer) constituted the PCR-reaction mix. All samples were applied in

duplicates at a final volume of 25 µl and added to a MicroAmp Optical 96-well

reaction plate with optical cover. A 7500 Fast Real-Time PCR (Applied

Biosystems) was used for amplification of the reaction mixture for 50 cycles,

with an annealing temperature of 58-60°C for primers and 68-72° for probes.

Quantification of target mRNA was acquired from the FAM dye layer, while the

VIC dye layer yielded the result for quantification of the 18S rRNA endogenous

control and the ROX dye layer was used for the passive reference used for

normalise for non-PCR-related fluctuations in fluorescence signal. An inter-

assay control RNA (i.e. unstimulated PBMC from a healthy individual) as well as

controls without template was included in all experiments. In paper III, sCTLA-

4 was not analysed for optimum stimulation period, due to limited amount of

study material.

Calculation of relative quantification values of mRNA gene expression was done

from the accurate cycle threshold (CT) according to the manufacturer’s

description (protocol P/N 4304671, Perkin Elmer). A threshold line was set in

the exponential phase of the amplification, the accurate CT value represents the

cycle where the increase in fluorescence in both dye layers in the assay have

passed the threshold. A low CT value represents a high mRNA expression and

vice versa. The CT value for rRNA (VIC layer) was subtracted from the CT value

of the immunological marker (FAM layer) to calculate Δ-CT for the calibrator

and samples in each T-reg associated marker gene expression assay. Δ-CT values

for duplicate wells of the calibrator sample for each marker were averaged

(spontaneous expression). The average delta (calibrator) value was subtracted

~ 52~

from the Δ-CT values of samples to calculate their ΔΔ-CT value. The ΔΔ-CT value

for duplicate wells of each sample was averaged. This operation normalises the

number of target mRNA molecules to the number of rRNA molecules. Relative

quantification values were obtained after calculating the 2-averaged ΔΔ-CT.

Enzyme linked immunosorbent assay (ELISA) (paper III)

Secretion of the cytokine TGF-β1 in cell supernatants were analysed in paper III

with Quantikine human TGF-β1 ELISA (R&D Systems, Abingdon, UK) according

to the manufacturer’s recommendation. Briefly, TGF-β1 in cell supernatants

were activated by addition of hydrochloric acid (HCl), diluted and incubated for

2 hours at RT. After several wash-steps, with wash buffer (included in the kit),

TGF-β1 conjugate was added, followed by incubation for 2 hours at RT. After

additional wash steps, as previously, substrate solution (included in the kit) was

added and incubated in darkness for 30 minutes at RT. Stop solution (included

in the kit) was added and the plate was read within 30 minutes at OD 450 nm.

The range of the standard curve was 31.2-2000 pg/ml, and a limited number of

samples had a concentration below the detection limit of the ELISA.

Flow cytometry

In paper V, flow cytometry was used to analyse both extra- and intracellular

expression of markers associated to Treg cells in whole blood. Staining was

performed within 24 hours from blood sampling. Blood was incubated for 15

minutes in darkness with extracellular antibodies associated to Treg cells;

fluorescein isothiocyanate (FITC)-conjugated CD39 (Biolegend, San Diego,

California, USA), peridinin-chlorophyll proteins-cyanin5.5 (PerCP-Cy5.5)-

conjugated CD45RA (Biolegend), PE-Cy7-conjugated CD25 (Biolegend),

allophycocyanin (APC)-conjugated CD127 (BD Biosciences, San José, California,

USA) and APC-Cy7-conjugated CD4 (Biolegend), followed by wash in PBS

~ 53~

(Medicago AB, Uppsala, Sweden) supplemented with 0,5 % bovine serum

albumin (BSA) (Sigma-Aldrich, St Louis, Missouri, USA) and centrifugation at

500 g for 5 minutes. After removal of cell supernatant with pasteur pipette, cells

were resuspended and treated with Fixation/Permeabilization buffer

(eBioscience, San Diego, California, USA) for 30 minutes at 4°C in darkness,

followed by centrifugation for 10 minutes at 500 g. After another wash step, cells

were resuspended in Permeabilization buffer (eBioscience) and intra cellular

PE-conjugated FOXP3 antibody was added. After 30 minutes of incubation in

darkness at RT cells were washed with PBS containing 0.5% BSA and

centrifuged for another 5 minutes at 500 g. After removal of cell supernatant,

cells were resuspended in PBS containing 0.5% BSA and kept at 4°C until

analysis.

Gating strategy and analysis of flow cytometric data

Flow cytometric data were analyzed with Kaluza 1.1 (Beckman Coulter,

Manchester, UK), and all analyses were blinded. Lymphocytes were gated based

on forward scatter (FSC) and side scatter (SSC). Initially, CD4+ cells were gated

(figure 7a), followed by gating of CD25+ cells (figure 7b). The CD4+ CD25+

population was either used in total (figure 7c) or after gating of the 2% highest

in this population, termed CD4+ CD25high (figure 7d). Results were either

expressed as frequency of expressed markers (%) or as MFI, which is equivalent

to the amount of receptors on the cell.

~ 54~

Figure 7: Gating strategy for a: Lymphocyte population, b: CD4+ population, c: Total CD4+ CD25+

population, d: Strict CD4+ CD25high population

Statistical analyses

As the data were not normally distributed, non-parametric statistical analyses,

corrected for ties, were used throughout the entire thesis. Three or more groups

were compared using Kruskal-Wallis, while Mann-Whitney U-test was used for

comparison between two groups. In paper III Wilcoxon signed rank test was

employed for paired observation for fresh versus cryopreserved PBMC and 48

hours versus 96 hours respectively. Correlation between mRNA expression and

Fig 7a Fig 7b

Fig 7c Fig 7d

~ 55~

protein levels was calculated by a bivariate, two-tailed Spearson correlation test.

A probability level of <0.05 was considered statistically significant, while a

probability level <0.1 was regarded a trend. In paper I calculations were

performed using the statistical package STATVIEW 5.0.1 for Macintosh (Abacus

Concepts Inc., Berkely, CA, USA), for paper 2 and 3 calculations were performed

using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA) and for paper 4 and 5

PASW Statistics 18.0 for Windows (SPSS Inc., Chicago, IL, USA).

Ethics

Collection of blood samples, SPT and questionnaires included in the study was

performed after informed consent from all parents or responsible guardians, or

the participant if he/she was over 18 years of age. The studies were approved by

the Ethics Committee of the Faculty of Health Sciences, Linköping University,

Sweden.

~ 57~

RESULTS AND DISCUSSION

During an immune response several different cells are involved e.g. T-, B-, Treg-,

NK and Tc-cells. These immunological cells secrete e.g. cytokines and

chemokines and express different immune markers that can be detected by

immunological methods such as Luminex technique, ELIspot, flow cytometry

and real-time RT-PCR. We have evaluated both secretion of cytokines and

chemokines, as well as markers associated with different cells in order to

evaluate the immune response in children with T1D, celiac disease, allergy, or a

combination of two of these diseases.

Children with T1D showed a decreased spontaneous IL-15 secretion and a

decreased spontaneous and PHA-induced expression of FOXP3

The spontaneous secretion of IL-15 was decreased in children with T1D in

comparison to children with allergy, combinations of T1D and celiac disease,

T1D and allergy, celiac disease and allergy as well as reference children (figure

8), when evaluated with Luminex-technique (paper II). Our results are in

contrast to a previous report showing that levels of IL-15 are elevated in serum

of T1D-patients [66]. This difference may be due to different methods, i.e. the

use of PBMC versus serum. Kuczynski et. al. [66] further found that high serum

levels of IL-15 are correlated to a high hemoglobin A1c (HbA1c). In NK-cell

depleted NOD-mice IL-15 has a protective role against T1D-development, and

IL-15 was found to both increase the suppressive effect of CD4+ CD25+ Tregs and

the FOXP3-expression in these cells [67]. Administration of a human IL-15-IgG-

2b recombinant cytokine to NOD-mice reduced the cumulative incidence of

diabetes, an effect that might be due to the anti-apoptotic effects of IL-15 [152].

Additionally, treatment with IL-15 fusion protein in combination with

~ 58~

rapamycin and IL-2 fusion protein stopped the progress of the autoimmune

destruction of β-cells and restored euglycemia [153].

Figure 8: Spontaneous secretion of IL-15 was decreased in peripheral blood mononuclear cells

(PBMC) from children with T1D as well as celiac disease, in comparison to children with allergy,

combinations of T1D and CD, T1D and AD and reference children. * p<0.05, ** 0.01≤p ≤0.001.

IL=Interleukin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease, Ref=Reference.

There is no clear picture regarding FOXP3-expression in children with T1D.

When analyzing FOXP3+ cells with flow cytometry no differences were seen in

frequency of FOXP3+ Treg either in patients with T1D, both recent-onset and

established T1D, their first-degree relatives or healthy subjects [69].

Additionally, Tiitanen et. al. [100] did not find any up-regulation of FOXP3-

expressing cells in mucosa from small intestine from patients with T1D.

However the percentage of intestinal CD4+ CD25+ FOXP3+ CD127- has recently

*

*

*

*

**

**

**

*

Fig 8

~ 59~

been shown to be decreased in T1D patients in comparison to healthy subjects

and individuals with celiac disease [72]. Dendritic cells from lamina propria in

T1D patients were further found to be unable to convert CD4+ CD25- cells into

CD4+ CD25+ FOXP3+ CD127- Treg cells [72]. However, this group of T1D

consisted of individuals with both T1D as a single disease and patients with T1D

and celiac disease in combination [72]. Further, effector T-cells in diabetic

individuals have been shown to be insensitive to regulation by induced CD4+

FOXP3+ cells, as well as nTreg direct isolated from blood, suggesting a defect

not in the Treg population but defects in effector T-cells instead [154]. Recently

Han et. al. [155] reported that patients with long-term T1D had a decreased fold-

change of FOXP3 mRNA-expression in comparison to both newly diagnosed

T1D-patients and healthy controls. We found that spontaneous mRNA-

expression was decreased in children with T1D in comparison to children with

celiac disease (figure 9a). Further, PHA-induced mRNA-expression of FOXP3

was reduced in children with T1D in comparison to children with celiac disease

and children with combination of T1D and celiac disease (figure 9b). Given the

diverging results from several studies regarding FOXP3-levels in T1D patients

it´s hard to draw an unambiguous conclusion, and obviously there is a need for

more research concerning this matter.

~ 60~

Figure 9: Spontaneous FOXP3 mRNA-expression was decreased in children with T1D in

comparison to children with celiac disease, as well as PHA-induced mRNA expression of FOXP3,

in comparison to children with celiac disease and combinations of T1D and celiac disease. In

contrast was the spontaneous and PHA-induced mRNA expression of FOXP3 increased in

children with celiac disease in comparison to reference. * p<0.05, ** 0.01≤p ≤0.001.

FOXP3=Forkhead box protein 3, PHA=Phytohaemagglutinin, T1D=Type 1 diabetes, CD=Celiac

disease, Ref=Reference.

In conclusion:

11.0

13.5

16.0

Spo

nt.

FO

XP

3 m

RN

Aex

pr.

(del

ta C

t)

T1D CD

T1D

+C

D Re

f

1.0

3.5

6.0

8.5

*

*

PH

A in

d. F

OX

P3

mR

NA

exp

r. (d

elt

a C

t)

T1D

CD

T1D

+C

D

Re

f

1.0

3.5

6.0

8.5

11.0

13.5

16.0

**

*

*Fig 9a Fig 9b

Type 1 diabetes

FOXP3

FOXP3

IL-15

PHA

Spontaneous

~ 61~

Children with celiac disease showed decreased spontaneous IL-4 and IL-

15 secretion but an increased expression of spontaneous, insulin-, gluten-

and PHA-induced FOXP3

Celiac disease has a Th1-associated immune response, reported previously by

several groups [93-95]. Interferon-γ is shown to modify epithelial barrier

function through a modification of tight junction function altering the

permeability of the epithelium [156], and expression of IFN-γ mRNA in mucosa

from patients with celiac disease was significantly increased in comparison to

healthy subjects [157]. Further IFN-γ mRNA-expression in intraepithelial

lymphocytes (IEL) was increased in three groups of children with celiac disease

(untreated, on GFD or challenged) in comparison to control subjects [94].

Further, expression of IFN-γ mRNA, but not IL-4 mRNA, was increased in

mucosa from patients with celiac disease in comparison to controls [95].

Secretion of IL-4 in celiac disease seems to be associated with disease activity, as

children with a treated celiac disease secreted no IL-4, while untreated patients

showed an increased secretion of IL-4 in peripheral leucocytes [93]. Still, serum

levels of IL-4 was shown to be elevated in children with untreated celiac disease

and those on GFD with antibodies against tTG, in comparison to children on

GFD without tTG antibodies or controls [158]. We found that children with

celiac disease showed a decreased IL-secretion assessed with ELIspot-technique

(paper I), in concordance with a Th1-profile which is the most dominant feature

in celiac disease patient (figure 10). The difference between reports showing an

increased or a decreased IL-4 secretion is probably due to different methods and

matrices, or difference in disease process, i.e. untreated versus treated.

~ 62~

Figure 10: Spontaneous secretion of IL- was decreased in children with celiac disease in

comparison to children with T1D, allergy and combined T1D and allergy. * p<0.05, ** 0.01≤p

≤0.001 *** p<0.001. IL=Interleukin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease,

Ref=Reference

Interleukin-15 is involved in the disease process in celiac disease, as

demonstrated in biopsies from small intestine in patients with celiac disease,

both treated and untreated, as well as healthy controls [97]. Induced IL-15

epithelial modulations were noted in patients with celiac disease, especially

untreated, but not in controls, and this modification could be inhibited by anti-

IL-15-antibodies [97]. Further, IL-15 was reported to be overexpressed both in

lamina propria and the intestinal epithelium in patients with untreated celiac

disease, including refractory celiac sprue, however not in treated patients or

controls [98]. Interleukin-15 has also been reported to have effects on celiac

disease process in mice. DePaolo et. al. [99] reported that when transgenic mice,

constitutively expressing IL-15, were stimulated with OVA together with retinoic

acid Treg cells were inhibited, a Th1-response via IFN-γ was induced and the

IL-4

[sp

ots

/10

0.0

00

PB

MC

]

Fig 10

0

10

20

30

40

50

60

70A

D

CD

T1

D

CD

+ A

T1

D +

AD

T1

D +

CD

Ref

**

***

** ***

~ 63~

secretion of the pro-inflammatory-associated cytokines IL-12p70 and IL-23 was

increased. Taken together, results from human models show that IL-15 is

important in development of celiac disease. This is especially noted in untreated

patients, which is in line with our findings showing that IL-15 is spontaneously

decreased in children with celiac disease (figure 8), as almost all children in our

cohort have been on GFD for at least one month and in most cases at least one

year (paper II).

In paper IV we examined FOXP3 mRNA-expression in children with T1D, celiac

disease, combination of T1D and celiac disease as well as reference children. We

found that children with celiac disease showed an increased spontaneous (figure

9a) and insulin- (figure 11a), gluten- (figure 11b) and PHA-induced (figure 9b)

mRNA-expression of FOXP3, in comparison to reference children. This is in

contrast to several studies showing an increased secretion or mRNA-expression

of FOXP3 mainly in individuals with an untreated disease, as the children in this

cohort in almost all cases have a treated celiac disease (on GFD for at least one

month). In a study by Tiittanen et. al. [100], FOXP3 intestinal mRNA-expression

was increased in children with an untreated celiac disease in comparison to

controls (without autoimmune diseases) and the same pattern was found when

studying FOXP3+ cells. This was further supported by Frisullo et. al [101] who

found both a higher percentage of circulating CD4+ CD25+ FOXP3+ cells and an

increased FOXP3 expression in CD4+ CD25+ T-cells in adults with untreated

celiac disease in comparison to adults with treated celiac disease. Additionally,

untreated adults show a higher density of CD4+ CD25+ FOXP3+ cells in intestine

biopsies in comparison to treated adults and controls without celiac disease,

and treated adults react with an expansion of FOXP3 after stimulation with

gliadin [102]. This difference in results probably is due to use of different

methods and biomaterials, and also differences in age of studied subjects.

~ 64~

Several studies have focused on site in the intestine, and it is likely that PBMC

do not fully reflect what happens in the gut. When separating PBMC with Ficoll

Paque, 95% of the cells are PBMC, this population consists of lymphocytes (T

and B), monocytes and NK-cells [3]. Even though frequencies of FOXP3+ cells

have been shown to be age-independent [69, 159], there might be an alteration

at mRNA-levels, as seen for IFN-γ mRNA being increased with age [155].

Figure 11: mRNA-expression of FOXP3 was increased in children with celiac disease in

comparison to reference after a) insulin stimulation or b) gluten stimulation. * p<0.05.

FOXP3=Forkhead box protein 3, T1D=Type 1 diabetes, CD=Celiac disease, Ref=Reference

In conclusion:

Glu

ten

ind

. FO

XP

3 m

RN

Ae

xpr.

(de

lta

Ct)

T1D

CD

T1D

+ C

D

REF

1.0

3.5

6.0

8.5

11.0

13.5

16.0

*

T1D

CD

REF

1.0

3.5

6.0

8.5

11.0

13.5

16.0

18.5

Insu

lin

ind

. FO

XP

3 e

xpr.

(de

lta

Ct)

11a *p=0.057

11b

Celiacdisease

FOXP3 Spontaneous, insulin, gluten and PHA

IL-15

IL-4Spontaneous

~ 65~

Children with allergy showed an increased birch- and cat-induced IL-4

secretion, an increased spontaneous IFN-γ, IL-10 and IL-12 secretion

Allergy and asthma are diseases with a Th2-deviated immune response, with

secretion of e.g. IL-4 that drives B-cells to produce IgE. Th2-associated cells have

also been demonstrated in the airways of individuals with atopic asthma [118,

160]. This was confirmed in paper I, where allergic children responded with an

increased IL-4 secretion after stimulation with both birch (figure 12a) and cat

(figure 12b), studied by ELIspot-technique.

Figure 12: Secretion of IL-4 was increased in allergic children after a) birch stimulation, in

comparison to children with T1D, celiac disease, combinations of celiac disease and allergy, T1D

and celiac disease and reference or b) cat stimulation, in comparison to children with T1D, celiac

disease, celiac disease and allergy in combination and reference. * p<0.05 ** 0.01≤p ≤0.001.

IL=Interleukin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease, Ref=Reference

Fig 12a

-40

-20

0

20

40

60

80

100

IL-4

by

bir

ch [

spo

ts/1

00

00

0 P

BM

C]

-40

-20

0

20

40

60

80

100

120

140

160

IL-4

by c

at

[sp

ots

/100 0

00 P

BM

C]

**

**

*

*

*

**

*

*

*

AD

CD

T1D

CD

+A

D

T1

D +

AD

T1

D +

CD

Ref

AD

CD

T1

D

CD

+A

D

T1D

+A

D

T1

D +

CD

Ref

Fig 12b

~ 66~

In atopic individuals cells producing IFN-γ have been shown to be decreased in

CD4+ cells, but not in CD8+ cells [119], and apoptosis of IFN-γ-producing cells

has been shown to be increased in atopic individuals [161]. However, there are

conflicting results, as IFN-γ, a Th1-associated cytokine, has been proposed to be

involved in asthma and atopy, in particular in CD8+ cells. Further, it has been

shown that the percentage of IFN-γ+ cells is increased in BAL fluid in patients

with atopic asthma, compared to atopic patients without asthma as well as

healthy controls [162]. Expression of IFN-γ mRNA in sputum has been

demonstrated to be increased in patients with moderate to severe asthma

compared to mild asthma, but mRNA expression of IFN-γ showed to be

decreased in allergic patients compared to non-allergic patients in the same

study [163]. These results were further supported by Shannon e. al. [164],

showing an increased IFN-γ and IL-8 expression in subepithelia in patients with

severe asthma compared to moderate asthma. Our data are in line with this

observation, showing an increased spontaneous IFN-γ secretion in children with

allergy, studied by both ELIspot (figure 13a) (paper I) and Luminex technique

(figure 13b) (paper II).

~ 67~

Figure 13: Spontaneous secretion of IFN-γ was increased in children with allergy in comparison to

almost all other groups with a)ELIspot-technique or b) Luminex-technique. * p<0.05 ** 0.01≤p

≤0.001. IFN=Interferon,, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease,

Ref=Reference

Interleukin-12 is another Th1-associated cytokine, shown to have an inhibitory

effect on IL-4-induced IgE-production in human PBMC [120] and also an

inhibition of airway hyperreactivity, inflammation and decrease of Th2-

asscociated cytokines in murine models of allergic asthma [121, 165]. Blocking IL-

12 in a murine model of experimental asthma has been reported to have

differential effects depending on when IL-12 is inhibited; blockade of IL-12

during the sensitization phase intensifies the allergic airway inflammation,

while blockade during the challenge phase instead diminishes the Th2-airway

inflammation [166]. In contrast to this we found that allergic children had an

increased spontaneous IL-12 secretion (figure 14). This is a difference that might

be due to use of different methods and cell types as we study secretion in

human PBMC, while some studies are performed in murine airways, with other

expressed cell types (paper II).

7338

*

**

****

*IF

N-g

[sp

ots

/100.0

00 P

BM

C]

0

50

100

150

200

250

300

350

400

450 *

**

*

*

*

Fig 13a

Fig 13a

AD

CD

T1D

CD

+A

D

T1D

+A

D

T1D

+C

D

Ref

~ 68~

Figure 14: Spontaneous secretion of IL-12 was increased in children with allergy in comparison to

combination of T1D and allergy and reference. * p<0.05 ** 0.01≤p ≤0.001. IL=Interleukin,

T1D=Type 1 diabetes, CD=Celiac disease, AD=allergic disease, Ref=Reference

Interleukin-10 is a cytokine produced by monocytes, B-cells, both Th1- and Th2-

cells, and to some extent also NK-cells [167, 168]. They are secreted by TR1-cells,

and polymorphisms in IL-10 genes have been reported to be associated to both

atopic and non-atopic asthma in children [40, 169]. Interleukin-10 has been

reported by several groups to be instrumental in the use of specific

immunotherapy (SIT). Grass pollen SIT significantly increases IL-10 in patients

undergoing grass pollen immune therapy in comparison to untreated atopic

controls [170], IL-10 has also been shown to be secreted by both specific T-cells

and antigen presenting cells after high doses of bee venom [123]. Monocytes

secreting IL-10 are increased in atopic patients, in comparison to healthy

controls [122]. We found that allergic children spontaneously secreted higher

200114

*

*

***

**

Fig 14

~ 69~

levels of IL-10 in comparison to all the other studied groups (figure 15), which is

in line with reports from other groups (paper III).

Figure 15: Spontaneous secretion of IL-10 was increased in children with allergy in comparison to

all other studied groups. * p<0.05 ** 0.01≤p ≤0.001. IL=Interleukin, T1D=Type 1 diabetes,

CD=Celiac disease, AD=Allergic disease, Ref=Reference

In conclusion:

**

*

****

****

Fig 15

Allergy

IL-4 Birch, cat

IL-10

IFN-γ

Spontaneous

~ 70~

Children with a combination of T1D and celiac disease showed a

decreased inhalant- and food-antigen-induced IFN-γ secretion, however

an increased Treg-associated response

In paper I we found that children with combination of T1D and celiac disease

showed a decreased secretion of IFN-γ after stimulation with βLG (figure 16a),

OVA (figure 16b), gluten (figure 16c), birch (figure 16d) and cat (figure 16e). As

far as we know, this has not been described previously. It has previously been

suggested that the immune system in the gut is involved in the development of

T1D. Expression of HLA-DR, HLA-DP and adhesion molecules has shown an

increased expression in T1D patients with normal mucosa, and in lamina propria

IL-1α+ and IL-4+ cells were expressed to great extent [129]. Gliadin challenge

among T1D patients give to hand that subsets of these patients show an

immunological response to gliadin with lymphocyte recruitment [126]. Gliadin

has also recently been reported to induce moderate enteropathy, and barrier

dysfunction in NOD-DQ8 mice, however insulitis was only induced after

depletion of CD25+ FOXP3+ T cells [131]. Additionally, as reviewed in [127],

gliadin is considered a part of the pathogenesis in both T1D and celiac disease,

which has effects on intestinal permeability and changes in tight junction

dynamics, through the binding of gliadin to CXCR3. All together this implies a

role of the immune system in the gut in both T1D and celiac disease that might

affect oral tolerance. However, this is a complex network and our results might

reflect a suppression of the immune response when combining two Th1-

associated diseases.

~ 71~

Figure 16: Secretion of IFN-γ was decreased in children with combination of T1D and celiac

disease a) after stimulation with βLG, in comparison to children with T1D, celiac disease,

combination of T1D and allergy and reference, and b) after stimulation with OVA in comparison

to children with T1D, celiac disease, allergy, combination of celiac disease and allergy and

reference, and c) after stimulation with gluten in comparison to reference children, and d) after

stimulation with birch in comparison to children with T1D, allergy, combination of T1D and

allergy and reference, and e) after stimulation with cat in comparison to children with T1D,

combination of T1D and allergy and reference. * p<0.05 ** 0.01≤p ≤0.001. IFN=Interferon,

βLG=Betalactoglobulin, OVA=Ovalbumin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic

disease, Ref=Reference.

IFN

-gb

y b

LG

[sp

ots

/100.0

00 P

BM

C]

****

*

*

-150-100-50

050

100150200250300350

IFN

-gb

y O

VA

[sp

ots

/10

0.0

00

PB

MC

]

*

*

**

**

**

-100

-50

0

50

100

150

200

-125-100-75-50-25

0255075

100125

5

IFN

-gb

y g

lute

n [

spo

ts/1

00

.00

0 P

BM

C]

*

AD

CD

T1

D

CD

+A

D

T1

D +

AD

T1

D +

CD

Ref

AD

CD

T1D

CD

+A

D

T1D

+A

D

T1

D +

CD

Ref

AD

CD

T1D

CD

+A

D

T1D

+A

D

T1

D +

CD

Ref

Fig 16a Fig 16b Fig 16a

IFN

-gb

y b

irch

[sp

ots

/10

0 0

00 P

BM

C] **

*

*

IFN

-gb

y c

at

[sp

ots

/10

0 0

00 P

BM

C] *

**

*

*

-200

-100

0

100

200

300

400

500

-200

-100

0

100

200

300

400

500

AD

CD

T1D

CD

+A

D

T1D

+A

D

T1

D+

CD

Ref

AD

CD

T1D

CD

+A

D

T1D

+A

D

T1

D+

CD

Ref

Fig 16d Fig 16e

~ 72~

In contrast to a suppressed Th1-response in combined T1D and celiac disease,

we found that these children have an increased expression of the Treg-

associated markers FOXP3 (figure 17a) and CD39 (figure 17b), and decreased

expression of CD127 (figure 17c-e) and CD45RA (figure 17d-e), both negatively

associated to Treg cells (paper V). First-degree relatives to T1D-patients have

been shown to have decreased percentage of CD4+ CD25+ CD127- cells in

comparison to healthy controls, and a tendency of decrease of CD4+ CD25+

FOXP3+ cells in comparison to healthy individuals [71]. In a study by Badami et.

al. [72], T1D patients were found to have a decreased percentage of CD4+ CD25+

FOXP3+ CD127- intestinal cells in comparison to healthy individuals and subjects

with celiac disease. However 5/10 T1D patients had additional celiac disease but

the difference remained after a division of the T1D-group into one with

exclusively T1D and one with combination of T1D and celiac disease [72].

Further did Badami et. al. [72] compare percentage of CD4+ CD25+ FOXP3+

CD127- intestinal cells to percentage of CD4+ CD25+ FOXP3+ CD127- in PBMC,

and found that the difference disappeared, which is in line with Brusko et. al.

[69], but in contrast to our findings.

In small-bowel mucosa from children with celiac disease, combination of T1D

and celiac disease and controls mRNA levels of FOXP3 were reported to be

increased in children with combined T1D and celiac disease in comparison to

both subjects with celiac disease and healthy controls [132]. Additionally, the

number of FOXP3+ cells were increased after immunohistochemical staining in

children with combination of T1D and celiac disease in relation to controls, and

the same was seen for children with celiac disease alone in comparison to

controls [132]. Our findings are unique in respect of studies in Treg-associated

markers in children with combination of T1D and celiac disease, and they are

indicating that this combination increases the Treg population, especially in

comparison to children with solely T1D or celiac disease.

~ 73~

Figure 17a: Increased frequency of FOXP3 cells in the strict CD4+ CD25high population, in children

with combination of T1D and celiac disease, in comparison to children with exclusively T1D.

Figure 17b: Increased MFI of FOXP3 the strict CD4+ CD25high population, on comparison to

children with exclusively celiac disease.

Figure 17: Decreased MFI of CD127 in total CD4+ CD25+ population, in comparison to children

with exclusively T1D.

Figure 17d: Decreased CD127 and CD45RA the strict CD4+ CD25high population, in children with

combination of T1D and celiac disease in comparison to children with T1D or celiac disease.

Figure 17e: Decreased CD127 and CD45RA the total CD4+ CD25+ population, in children with

combination of T1D and celiac disease in comparison to children with T1D or celiac disease.

* p<0.05, ** 0.01≤p ≤0.001. MFI=Median fluorescence intensity, FOXP3=Forkhead box protein 3,

T1D=Type 1 diabetes, CD=Celiac disease, Ref=Reference.

* **Fig 17bFig 17a

~ 74~

In conclusion:

Children with a combination of T1D and allergy showed an increased

PHA-induced IFN-γ and IL-4 secretion, as well as an increased birch-

induced secretion of several cytokines

In paper I we showed that children with a combination of T1D and allergy

showed an increased PHA-induced IFN-γ (figure 18a) and IL-4-response (figure

18b). This was in contrast to a study by Rachimel et. al. [136] who analyzed IFN-

γ, IL-2, IL-4 and IL-10 secretion with ELIspot and ELISA, after stimulation with

GAD, house dust mite, PHA, TT and anti-CD3 monoclonal antibody. They

found that children with a combination of T1D and asthma and children with

T1D as single disease had similar cytokine pattern [136]. However, children with

a combination of T1D and celiac disease had a decreased Th1/Th2 ratio in

comparison to diabetic subjects [136]. Only stimulation with house dust mite

induced an increased secretion of Th2 cytokines (IL-4 and IL-13) in children

with a combination of T1D and celiac disease, as well as the total (i.e. both

T1D + celiacdisease

IFN-γ βLG, OVA, gluten, birch and cat

FOXP3CD39

Frequencyand MFICD4+ CD25high andCD4+ CD25+

CD45RACD127

Frequencyand MFICD4+ CD25high andCD4+ CD25+

~ 75~

unstimulated and stimulated) secretion of IL-10 [136]. The difference to our

findings may be caused by differences in age, or the fact that the allergic

children in our cohort consisted of children with asthma, atopic dermatitis, food

allergy and allergic rhinoconjunctivis.

Further, Kainonen et. al. [137] published a study the same year, where they

analyzed the concentration of IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 with a

multiplexed flow cytometric assay in PBMC, after stimulation with anti-CD3 and

anti-CD28 in children with T1D, asthma, a combination of T1D and asthma and

healthy children as controls. They found an increased spontaneous secretion of

IFN-γ, TNF-α and IL-10 in children with combined T1D and asthma in

comparison to children with T1D, asthma or healthy children [137]. Stimulation

with CD3 and CD28 only increased IL-10 secretion in children with exclusively

T1D or asthma, and controls, but not in children with T1D and asthma in

combination [137]. The differences displayed between our findings and

Kainonens is probably due to different methods as well as the fact that PHA and

CD3 use different pathways for stimulation [171, 172].

~ 76~

Figure 18: An increased PHA-induced secretion of a) IFN-γ in children with T1D and allergy in

comparison to children with T1D, allergy, combinations of T1D and celiac disease, celiac disease

and allergy and reference, and b) IL-4 in children with T1D and allergy in comparison to all other

studied groups. * p<0.05, ** 0.01≤p ≤0.001 *** p<0.001. IFN=Interferon, IL=Interleukin,

PHA=Phytohaemagglutinin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease,

Ref=Reference

We also found that children with combination of T1D and allergy increased the

secretion of numerous cytokines (IL-2 (figure 19a), IL-4 (figure 19b), IL-5, IL-7

(figure 19c), IL-9 (figure 19d), IL-10, IL-12 (figure 19e), IL-15 (figure 19f), IL-17 and

CCL11) after stimulation with birch when analyzed with Luminex-technique

(paper II). Serum levels of IL-12 and IL-18 have been reported to be increased in

children with T1D and asthma in comparison to controls [138]. However LPS

decreases the IL-12 secretion in children with combined T1D and asthma in

comparison to children with T1D or asthma as only disease [138]. The study by

Rachmiel et. al. [138] differs from ours with respect to age-distribution of the

IFN

-g b

y P

HA

[sp

ots

/100

.000 P

BM

C]

IL-4

by

PH

A [

spo

ts/1

00

.00

0 P

BM

C]

*

*

***

*

**

*

*

*

*

*

**

*

0

100

200

300

400

500

600

700

800

900

0

100

200

300

400

500

600

700

800

900

AD

CD

T1

D

CD

+A

D

T1

D+

AD

T1

D+

CD

Ref

AD

CD

T1

D

CD

+A

D

T1

D+

AD

T1D

+C

D

Ref

Fig 18a Fig 18b

~ 77~

study group, different methods used and the fact that PHA and LPS use

different pathways for stimulation [171, 173].

Figure 19: Stimulation with birch increased a) IL-2 secretion in children with T1D and allergy in

combination in comparison to all other studied groups, and b) IL-4 secretion in children with

T1D and allergy in combination in comparison to children with allergy, combinations of T1D and

celiac disease, celiac disease and allergy and reference, and c) IL-7 secretion in children with

combination of T1D and allergy in comparison to all other groups, and d) IL-9 secretion in

children with T1D and allergy in comparison to all other groups, and e) IL-15 secretion in children

with T1D and allergy in combination in comparison to children with T1D, celiac disease, allergy

and combination of T1D and celiac disease, and f) secretion of IL-17 in children with combination

of T1D and allergy in comparison to children with T1D, allergy, combination of T1D celiac disease,

celiac disease and allergy and reference. * p<0.05, ** 0.01≤p ≤0.001 *** p<0.001. IL=Interleukin,

T1D=Type 1 diabetes, CD=Celiac disease, AD=allergy and Ref=Reference.

***

****

**

****

19a

**

*

*

*19b

***

*****

***

****

19c

*

***

**

****

**19d

**

**

19e

*****

***

**19f

~ 78~

In conclusion:

Children with a combination of celiac disease and allergy showed a

decreased spontaneous secretion of several cytokines

Interestingly, we found that children with a combination of celiac disease and

allergy showed a spontaneous decreased secretion of several cytokines (IL-1β,

IL-5, IL-6, IL-9 (figure 20a), IL-10 (figure 20b), IL-12 (figure 20c) and CCL3), a

result that, to the best of our knowledge, not yet has been reported by other

groups (paper II). Even though celiac disease is considered a Th1-deviated

disease, and allergy is considered a Th2-deviated disease, there are reports

showing that these diseases can coexist. In an Italian study, the prevalence of

celiac disease in atopics was significantly increased in comparison to the general

Italian population [140], which has been shown in a Finnish study as well, where

the cumulative incidence of asthma in children was increased in children with

celiac disease in comparison to children without this disease [141]. However, the

information of the immune response in children with celiac disease and allergy

is scarce, if not totally absent, which makes our results unique.

T1D + Allergy

IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17, CCL11

Birch

IL-4PHA

IFN-γ

~ 79~

Figure 20: Decreased spontaneous secretion of a) IL-5 in children with combination of celiac

disease and allergy in comparison to children with allergy, and b) IL-10 in children with

combination of celiac disease and allergy in comparison to children with T1D, allergy and

combination of T1D and allergy, and c) IL-12 in children with combination of celiac disease and

allergy in comparison to children with exclusively T1D or allergy. * p<0.05, ** 0.01≤p ≤0.001.

IL=Interleukin, T1D=Type 1 diabetes, CD=Celiac disease, AD=Allergic disease and Ref=Reference.

In conclusion:

**

*

****

****

Fig 20b 200114

*

*

***

**

Fig 20c

***

*

**

***

Fig 20a

Celiacdisease + Allergy

IL-1β, IL-5, IL-6, IL-9, IL-10, IL-12, CCL3

Spontaneous

~ 80~

Methodological data

In paper III we performed a methodological study in order to evaluate how

cryopreservation affects Treg-associated markers (FOXP3, TGF-β, CTLA-4 and

sCTLA-4), as well as an examination of suitable time, 48 or 96 hours, for

detection of mRNA-expression of those markers. We found that cryopreserved

PBMC retain a stable spontaneous mRNA-expression throughout the

cryopreservation, and stimulated TGF-β, CTLA-4 and sCTLA-4, as well as

secreted TGF-β remained stable (figure 21-23). Recently, our group has reported

that cryopreserved CD4+ CD25+ CD127lo/- cells from PBMC still are able to

expand after cryopreservation with a mean fold change of 144 and a median fold

change of 104 [174]. This is in line with a study by Venet et. al. [148] examining

how cryopreservation affects CD4+ CD25+ CD127- cells from HIV-infected

subjects. They found that even if isolation of PBMC with Ficoll decreased the

MFI of CD4+ cells in comparison to whole blood, no further decrease was seen

after cryopreservation [148]. Additionally, they found that CD4+ CD25+ CD127-

cells remained stable after cryopreservation both in comparison to fresh PBMC

and whole blood, and even if a slight increase of percentage of Treg was visible,

there was no statistically significant difference [148]. In contrast to the work by

Elkord [146], reporting a decrease in frequency of CD4+ FOXP+ cells in PBMC

from healthy donors, this difference is likely due to different experimental

setups, i.e. real-time RT-PCR versus flow cytometry or differences in the freezing

protocol, as described by Van Hemelen [147]. Further we found that a

stimulation period of 48 hours in general was most suitable for detection of

FOXP3, TGF-β and CTLA-4 mRNA (figure 24, 25). Spontaneous mRNA-

expression was decreased after a 96 hours stimulation period in comparison to

48 hours; the same was seen in PHA-induced FOXP3 and TGF-β mRNA-

expression, as well as for TT-induced FOXP3 mRNA-expression. This result is in

line with a study by Allen et. al. [175] reporting the percentage of expressed

~ 81~

FOXP3, CD25, CTLA-4, CD69 and glucocorticoid-induced tumor necrosis factor

receptor compared between non-regulatory T-effector cells expressing CD4 and

naturally occurring CD4+ CD25+ during seven days, showing that the expression

of FOXP3 increased during the first 72 hours, followed by a decline.

~ 82~

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on

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XP

3

TG

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CT

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pre

serv

ed

14

15 16 17 18

19

20

10 11 12

13

14

Fre

shC

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pre

serv

ed

15

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*

* *

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19

20 21 22 23 24 25 26

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shC

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ed

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~ 86~

SUMMARY AND CONCLUSION

Numerous cell types and cell populations are involved during an immune

response, in an intricate and balanced network. Occasionally this balance is

disturbed, leading to different diseases. One working model that tries to explain

this is the concept of Th1- and Th2-cells, commonly believed to be inhibit each

other. Further, cells with regulatory features, Tregs, are also important, as these

have the function of halting an ongoing immune response in order to avoid

harmful effects.

Type 1 diabetes is an autoimmune disease caused by the destruction of insulin-

producing β-cells in pancreas. The cause behind is still unknown, and as there is

yet no cure, individuals with T1D need a life-long treatment with insulin. Type I

diabetes is suggested to be of a Th1-deviated feature.

In celiac disease gluten, a protein present in major crops but dominant in wheat,

induces an immune response leading to villous atrophy and crypt hyperplasia in

the small intestine. The only cure for celiac disease is to exclude gluten from the

diet for the rest of life. From an immunological point-of-view, Th1-deviation has

been proposed in celiac disease as well.

Allergy has increased several-folds during the last 4-5 decades particularly in

affluent countries. Allergic disease affects different organs depending on the

specific allergen. A Th2-response is initiated after exposure to an allergen, which

in itself is not a threat but recognized by the immune system as something

harmful.

Patients with T1D or celiac disease have an increased risk to develop the other

disease, because of shared risk genes, and this is a more frequent combination,

than combining T1D and allergy or celiac disease and allergy. Due to the

paradigm of a balance between Th1 and Th2, the combination of one Th1-

~ 87~

deviated disease with one Th2-deviated seems unlikely, but is a reality. In this

thesis I examined the immune response in children with T1D, celiac disease,

allergy, or a combination of two of these diseases in relation to healthy children,

with focus on Th1, Th2 and Treg subsets of lymphocytes. Additionally, I

performed a methodological study in healthy adults, to examine the effect of

cryopreservation and optimal stimulation-period for Treg-associated markers.

Some of our results were expected with regard to previous reports on Th1- and

Th2-cells during different diseases. Studying children with one of these diseases

showed a decreased spontaneous Th2-response in children with celiac disease

and an increased Th2-response against birch and cat in allergic children.

However, allergic children displayed an elevated spontaneous Th1-response,

which could be due to a previously reported participation of IFN-γ in children

with asthma, as well as an increased IL-10 secretion, implying an increased

involvement of TR1 cells in children with allergy.

The most unique and interesting results in this thesis are those seen in children

with combinations of these diseases, as these combinations of diseases have not

been studied to a great extent previously. We found that the combination of

T1D and celiac disease decreased the Th1-response against both inhalation- and

foodantigens, but gave a more pronounced Treg-response. The combination of

T1D and allergy increased both Th1- and Th2-response against a general

stimulus, as well as in increased Th1-, Th2-, Treg- and pro-inflammatory

response against birch. However, the combination of celiac disease and allergy

instead decreased the spontaneous Th1-, Th2-, Treg- and pro-inflammatory

responses.

~ 88~

Thus, it actually seems that a combination of two Th1-associated diseases

suppresses the immune response and activates regulatory mechanisms, while

allergy shifts the immune response in different directions depending on which

disease it is combined with. Speculatively, the suppressive effect could be

caused by exhaustion of the immune cells from the massive pressure of two

immunological diseases, whereas the more pronounced Tr-regulatory activation

could be due to an attempt to compensate for dysfunction in the immune

response. Still, further investigation is needed to examine why the immune

response acts in diverging directions.

In the methodological study we found that Treg-associated markers are suitable

to use after cryopreservation, as there were no significant difference between

fresh and cryopreserved PBMC with regard to mRNA-expression of FOXP3,

CTLA-4, sCTLA-4 and TGF-β. Yet, it is important not to use fresh and

cryopreserved PBMC in the same study. Further is a stimulation period of 48

hours was found in general to be the most optimal for studying mRNA-

expression of Treg-associated markers.

In conclusion, the immune system is a complex network. The Th1-Th2 paradigm

is an over-simplification, but still a useful working model. Speculatively is it

possible that different subpopulations of lymphocytes are displayed in all

diseases, but at different levels and directions. Our results shed some light on

the thrilling interplays of the immune system, and hopefully it will have some

effect on disease prevention and treatment.

~ 89~

FUTURE PERSPECTIVES

This cohort is unique, and includes a satisfying number of children. There are

several things that can be studied in order to seek more clues regarding this

specific cohort. One remarkable result not included in the thesis, was seen in

allergic children, who reacted with a high Th2-response against insulin. Thus,

we analyzed insulin IgE, but did not receive any significant results. We have

searched the literature to find any possible explanation, with no result so far.

Numerous theories have been rejected, hence this is a question that needs to be

elicited more thoroughly.

As the immunological universe is expanding, other cell types, like Nk-cells and

Tc-cells could be considered to be analyzed. An ongoing study, in cooperation

with the county hospital Ryhov, Jönköping, not only increases the cohort, but

also introduces new laboratory protocols. The aim is to increase the cohort from

paper V, including all three diseases and perhaps even introduce a group

consisting of children with all three diseases. Hence, to elucidate how these

three immunological diseases interacts, this project is still ongoing.

~ 91~

ACKNOWLEDGEMENT

A thesis might be my personal, but without help I would not be here today, so I

would like end this thesis by expressing my sincere gratitude to all of you, who

helped me coming this far.

All the children participating in this study.

My main supervisor Maria Faresjö: Thank you for letting me be part of this, letting

me see the world from a researcher’s point of view. You are an ambitious woman,

and I am sure you will reach every goal you have in life.

Lennart Nilsson, my first co-supervisor: I think every PhD-student should have a

supervisor like you, especially during the last year. Thank you for being there for

me!

Karin Åkesson, my second co-supervisor: Your enthusiasm is marvelous, and I think

your energy is endless. Thank you for sharing some of it with me.

Karin Fälth-Magnusson: Your kindness is amazing and your knowledge is deep.

Thank you very much for helping me during these years!

Forum Scientium, the multidisciplinary research program at Linköping University,

for proving good possibilities for networking and cooperation across disciplines

within Life Sciences.

Peter Bang, present representative for Division of Pediatrics, and Johnny

Ludvigsson, former representative for Division of Pediatrics.

~ 92~

There are several individuals that make the life of a PhD-student much easier. Kicki

Helander “my” research nurse, besides taking excellent care of the families in this

study, you have always helped me and cheered me up whenever we have met. Anki

Gilmore-Ellis, you are an angel without wings! Maria Jenmalm, for interesting

research discussions. Madeleine Örlin, for your kindness. Florence Sjögren and Uno

Johansson for excellent technical assistance, and for answering every question a

million times without any sign of irritation. Kerstin, Håkan, Pia for taking care of

“every-day-life” at KEF and sharing your knowledge.

And all my wonderful friends, many of you former or present members of

Fikarummet™, the place where problems are solved and laughter are near. So here

goes, in random order: Jenny Walldén and Sara Tomičić, there were no others

choice for toast madams, I miss you both a lot, and admire your happiness and your

life-wisdom! Anna Rydén, what can I say? “I says hi?” ;). Thank you for making any

conference trip epic!

Ingela Johansson, next year we have to do some “backträning”! You are a really nice

person, and you are always welcome at “Camp VR”! Ammi Fornander, Gosia

Smolinska-Konefal, Martina Sandberg, Camilla Skoglund, Piiha-Lotta Jerevall,

Mimmi Persson, Anne Lahdenperä, Tina Bendrik and Kristina Briheim for being

helpful, kind and just lovely persons. Anna Lundberg, one of the best boxing

partners I have had, but also a very dear friend.

Present and former roomies: Stina “Shakira” Axelsson, you are caring and sweet,

I´m going to miss you! Kristina Warstedt, not only my roomie but also a very dear

friend. This would have been a much harder experience without you! Linda

Åkerman, you are kind, and you always, always go that extra mile to help someone.

Malin Fagerås, you are a really wise person, and kind of a role model.

~ 93~

Mikael Chéramy, Mikael Pihl, Rosaura Casas for being part of the coffee-drinkers,

and for giving me good laughter. All other personnel at KEF.

For my friends outside work; Karin, now I know that true friendship can be born at

F&S! Jessica, thank you for being there whenever I need it! Lasse B, for being so

happy and enthusiastic all the time! Lovely, lovely Sofi, you are dear to me!

Tills sist:

Kim: Alla dessa år, och nu är den klar! Jag tror banne mig att jag siktar Motala nu!

Tack.

Mina underbara syskon Eva, Åsa och Otto med familjer. Det här hade inte gått utan

erat stöd, erat peppande, allt bollande, all barnvakt, ja listan kan göras lång…

Magnus: Tack för allt. Det kanske inte är ”bara ord” trots allt?

Mamma Helle och pappa Sven: Bättre föräldrar finns inte. Ert ändlösa stöd, er tro på

mig, alla peppsamtal har burit mig framåt. Att kunna komma hem till nybadade

barn, städad lägenhet och färdiglagad mat, det ÄR verkligen en lyx, och har betytt

mer än jag kan säga. Tack blir så torftigt, men det är det enda jag kan komma med,

så, från djupet av mitt hjärta: Tack! ♥

Klara och Michael: Ni visar mig vad som är viktigt i livet.

This work was generously supported by grants from Medical Research Council of

South-East Sweden (FORSS), Swedish Child Diabetes Foundation

(Barndiabetesfonden), Medical Research Fund of the County of Östergötland, the

County council of Östergötland, Schelins Foundation, the Research Foundation of

the Swedish Asthma and Allergy association, the Lions Foundation and Knut and

Alice Wallenberg’s foundation.

~ 95~

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