Studies on Antigen-Presenting Cells in Type 1 Diabetes and Crohn’s Disease – with Special

Emphasis on Dendritic Cells

Janne K. Nieminen

Immune Response Unit Department of Vaccination and Immune Protection

National Institute for Health and Welfare Helsinki, Finland

and

Children’s Hospital University of Helsinki and Helsinki University Central Hospital

Helsinki, Finland

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Medicine of the University of Helsinki, for public examination in the Niilo Hallman Auditorium, Children’s

Hospital on 5th December 2013, at 12 noon.

Helsinki 2013

Supervisors Professor Outi Vaarala, MD, PhD Immune Response Unit Department of Vaccination and Immune Protection National Institute for Health and Welfare Helsinki, Finland

Professor Mikael Knip, MD, PhD Children’s Hospital University of Helsinki and Helsinki University Central Hospital Helsinki, Finland

Reviewers Docent Arno Hänninen, MD, PhD Department of Medical Microbiology and Immunology University of Turku Turku, Finland

Docent Marko Pesu, MD, PhD Institute of Biomedical Technology University of Tampere Tampere, Finland

Official opponent Professor Hemmo A. Drexhage, MD, PhD Department of Immunology Erasmus Medical Center Rotterdam, the Netherlands

ISBN 978-952-10-9555-9 (paperback) ISBN 978-952-10-9556-6 (PDF) http://ethesis.helsinki.fi

Unigrafia Helsinki 2013

To Anna, Teresa, and Ossi

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Abstract

Type 1 diabetes (T1D) is considered an autoimmune disease wherein autoreactive infiltrate of immune cells destroys the insulin-producing pancreatic β cells. Crohn’s disease (CD) is a chronic inflammatory bowel disease thought to result from an exaggerated immune response against the gut commensal microbiota. In this thesis, antigen-presenting cells (APCs), in particular dendritic cells (DCs), were investigated in these conditions. DCs play a central role in the induction of immunity. After being activated by certain signals, such as microbial stimuli, DCs carry out antigen presentation to T cells and license them to mount the immune response.

We found reduced numbers of both circulating myeloid (mDCs) and plasmacytoid DCs (pDCs) in children with recent-onset T1D. In a flowcytometric immunophenotypic characterization, decreased expression of C-C chemokine receptor 2 (CCR2) was observed in children with T1D, which was confirmed in a separate series of children using RT-qPCR. DCs from children with signs of β-cell autoimmunity (a combined cohort of children with and without overt diabetes) also showed a trend towards an enhanced IL-1β-induced NF-κB reactivity. Although no causality could be established, this phenomenon is possibly interrelated with the CCR2 reduction because CCR2 was downregulated in vitro by NF-κB-inducing agents. These alterations may have functional consequences on DC migration and DC-induced T-cell activation in T1D.

T cells from pediatric T1D patients showed elevated induction of IL-17, and the IL-17-producing cells corresponded to the typical Th17 phenotype. The expression of Foxp3, an important regulatory T cell (Treg) –related transcription factor, was also increased and correlated with IL-17 and RORC2 expression. This finding is of interest, as Treg phenotypic dysregulation and instability has been described in T1D and in experimental diabetes. Using human pancreatic islets and a mouse insulinoma cell line we confirmed our hypothesis that IL-17 has direct detrimental effects on pancreatic β cells.

In CD, alterations in the expression and activation of STAT1, and in particular STAT3, have been described. As these signaling molecules and transcription factors regulate DC function, we asked whether disturbed STAT3 and/or STAT1 signaling would also characterize DCs in CD patients. However, no universal alteration of cytokine-induced STAT3 or STAT1 phosphorylation was found by flow cytometry in patients’ DCs. Instead, a pDC-restricted attenuation of IL-6/STAT3 signaling emerged, and the IFN-α-induced STAT1 and STAT3 signaling responses were decreased in both pDCs and CD1c+ mDCs. In contrast, IL-10-induced STAT3 phosphorylation was enhanced in CD1c+ mDCs from CD patients. IL-6 treatment of healthy donor pDCs resulted in an upregulation of IL-10 in the cocultured naive CD4+ T cells and a decreased ratio of Th1 to Th2 cytokine signature. Hence, impaired IL-6/STAT3 signaling in CD patients’ pDCs may be connected to the well-established phenomena of enhanced IFN-γ responses and insufficient IL-10 activity in the CD-

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related inflammation. Type I IFNs, such as IFN-α, are involved in the regulation of DC functions. In their absence, considerable functional alterations have been described, such as an impaired uptake of antigen and lymphocyte activation by the DCs. The attenuated IFN-α-induced responses we observed in CD are therefore of functional interest and may play a role in CD-associated immune dysregulation. We also assessed the numbers of circulating DC subsets in CD and found that pDCs and BDCA3+ mDCs were decreased and counts negatively correlated with the endoscopic disease activity. It is not clear whether the decreased DC counts are secondary to the inflammation. However, this reduction may also be pathophysiologically relevant, given the known homeostasis-promoting functions of these subsets. On the other hand, CD1c+ mDCs were more activated in terms of CD40 expression, which is in line with earlier reports.

Last, we asked whether the well-established enhanced IL-17 activation in CD is supported by an exaggerated Th17 induction by DCs. Contrary to our hypothesis, we found attenuated DC-supernatant-mediated upregulation of T cell IL-17A expression by LPS-stimulated monocyte-derived DCs from patients. In patients’ DCs, LPS-induced transcription of IL-1β and IL-6, together with autophagy-related LC3 mRNA upregulation, was also decreased. These findings are in support for the hypothesis of an innate immune defect in CD.

In conclusion, our findings from both T1D and CD suggest dysregulated DC function as a part of the immunological background from which these diseases develop.

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Contents

Abstract ......................................................................................................................... 4

Abbreviations ............................................................................................................... 8

List of original publications ...................................................................................... 13

1 Introduction ............................................................................................................ 14

2 Review of the literature ......................................................................................... 16 2.1 Dendritic cells ................................................................................................... 16

2.1.1 DC classification and identification ........................................................... 17 2.1.1.1 Myeloid DCs ....................................................................................... 18 2.1.1.2 Plasmacytoid DCs ............................................................................... 19

2.1.2 DC activation and some elements of its regulation ................................... 20 2.1.2.1 NF-κB and IRF activation in DCs ...................................................... 21 2.1.2.2 Cytokine-induced signaling and JAK-STAT-pathways in DCs ......... 23

2.1.3 Antigen-presentation and T-cell activation by DCs ................................... 24 2.2 T cells ................................................................................................................ 25

2.2.1 CD4+ T-helper effector cells ...................................................................... 26 2.2.2 CD8+ cytotoxic T cells ............................................................................... 27 2.2.3 Regulatory T cells ...................................................................................... 28

2.3 Immunological tolerance - with special reference to DCs ................................ 29 2.4 Some aspects of DCs in gut-related immunoregulation ................................... 32 2.5 Type 1 diabetes ................................................................................................. 33

2.5.1 Diagnosis and classification of diabetes .................................................... 34 2.5.2 Pathogenesis of T1D .................................................................................. 35

2.5.2.1 T cells in T1D ..................................................................................... 37 2.5.2.2 DCs and monocyte/macrophages in autoimmune diabetes – lessons from animal models ................................................................ 38 2.5.2.3 DCs and monocyte/macrophages in human T1D ............................... 39

2.6 Crohn’s disease ................................................................................................. 41 2.6.1 Diagnosis and treatment of CD .................................................................. 41 2.6.2 Pathogenesis of CD .................................................................................... 42

2.6.2.1 CD and STAT3 ................................................................................... 43 2.6.2.2 CD and experimental intestinal inflammation – viewpoint of T cells ............................................................................................. 44 2.6.2.3 DCs in CD and in experimental intestinal inflammation .................... 46

3 Aims of the study .................................................................................................... 49

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4 Materials and methods .......................................................................................... 50 4.1 Study subjects .................................................................................................... 50 4.2 Laboratory methods ........................................................................................... 50

4.2.1 Isolation of T cells and APCs .................................................................... 50 4.2.2 Cell-culture experiments ............................................................................ 52 4.2.3 Flowcytometric analyses ............................................................................ 53 4.2.4 Cytokine and chemokine measurements from serum, plasma, and cell-culture supernatant samples ......................................................... 54 4.2.5 RT-qPCR ................................................................................................... 55 4.2.6 Determination of islet cell viability and apoptosis .................................... 55 4.2.7 Diabetes-associated autoantibodies ........................................................... 56 4.2.8 HLA genotyping ........................................................................................ 56

4.3 Statistical analyses ............................................................................................ 56 4.4 Ethical considerations ....................................................................................... 57

5 Results and discussion – Type 1 diabetes ............................................................. 58 5.1 Children with T1D show signs of enhanced Th17 immunity ........................... 58 5.2 IL-17 augments the inflammatory and apoptotic effects of proinflammatory cytokines in pancreatic islet cells in vitro ............................. 60 5.3 Unaltered IL-1β and IL-6 transcription in APCs from diabetic children ......... 61 5.4 Numbers of circulating CD1c+ mDCs and pDCs are decreased in recent-onset pediatric T1D ................................................................................ 63 5.5 Expression of CC-chemokine receptor 2 (CCR2) and activation of NF-κB pathway in peripheral blood DCs in T1D ............................................. 64

6 Results and discussion – Crohn’s disease ............................................................ 69 6.1 Decreased numbers of peripheral blood pDCs and BDCA3+ mDCs and changes in DC phenotype in CD ....................................................................... 69 6.2 Altered cytokine-induced STAT1 and STAT3 phosphorylation in DCs from CD patients ............................................................................................... 71 6.3 Monocyte-derived DCs from CD patients have a decreased potential to activate Th17 responses in allogeneic memory cells by soluble mediators ...... 75

7 Conclusions ............................................................................................................. 80

8 Epilogue – general discussion ............................................................................... 81

9 Acknowledgements ................................................................................................ 85

10 References ............................................................................................................. 87

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Abbreviations

2-ME 2-mercaptoethanol AAb autoantibody Ab antibody Ag antigen AIRE autoimmune regulator APC antigen-presenting cell ATG16L1 autophagy-related protein 16-like 1 AU arbitrary unit BB Biobreeding (rat) BCL-2 B cell lymphoma 2 BDCA blood dendritic cell antigen BMDC bone marrow-derived dendritic cell BSA bovine serum albumin C5a complement component 5a cAMP cyclic adenosine monophosphate CCL chemokine (C-C motif) ligand CCR C-C chemokine receptor CD1 cluster of differentiation CD2 Crohn’s disease CDAI Crohn’s disease activity index cDC conventional dendritic cell COX-2 cyclooxygenase 2 CpG CpG-rich hypomethylated oligodeoxynucleotides CT computed tomography CTL cytotoxic T lymphocyte CTLA-4 cytotoxic T lymphocyte antigen 4 CX3CR1 CX3C chemokine receptor 1 CXCL chemokine (C-X-C motif) ligand DC dendritic cell DC-sign dendritic cell-specific intercellular adhesion molecule-3-grabbing non-

integrin DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid FAM 6-carboxyfluorescein FCS fetal calf serum FGL2 fibrinogen-like protein 2 Flt3 fms-like tyrosine kinase 3 Foxp3 forkhead box 3 G-CSF granulocyte colony-stimulating factor GAD glutamate decarboxylase

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GADA antibodies against glutamate decarboxylase GALT gut-associated lymphoid tissue GFP green fluorescent protein GM-CSF granulocyte-macrophage colony-stimulating factor GWAS genome-wide association study HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEV high endothelial venule HLA human leukocyte antigen HO Hoechst 33342 IA-2 islet antigen 2 IA-2A antibodies against islet antigen 2 IAA antibodies against insulin IBD inflammatory bowel disease ICA islet cell antibodies ICOS inducible costimulator IDO indoleamine 2,3-dioxygenase IEC intestinal epithelial cell IFN interferon Ig immunoglobulin IKK IκB kinase IL interleukin IL-10R interleukin-10 receptor IL-12R interleukin-12 receptor IL-17RA interleukin-17 receptor A IL-17RC interleukin-17 receptor C IL-18R interleukin-18 receptor IL-1R interleukin-1 receptor IL-2Rα interleukin-2 receptor α-chain ILT4 immunoglobulin-like transcript 4 IPEX immune dysfunction, polyendocrinopathy, enteropathy, X-linked IRF interferon regulatory factor iTreg induced regulatory T cell IκB inhibitors of κB JAK Janus kinase LADA latent autoimmune diabetes in adults LAG-3 lymphocyte activation gene 3 LC3 microtubule-associated protein 1A/1B-light chain 3 LGP2 laboratory of genetics and physiology 2 Lin lineage LN lymph node LP lamina propria LPS lipopolysaccharide mAb monoclonal antibody

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Mal MyD88 adapter-like protein MCP monocyte chemoattractant protein MDA5 melanoma differentiation-associated gene 5 mDC myeloid dendritic cell MDP muramyl dipeptide MFI median fluorescence intensity MHC major histocompatibility complex MLN mesenterial lymph node MLR mixed leukocyte reaction MNV murine norovirus moDC monocyte-derived dendritic cell MODY maturity-onset diabetes of the young MRI magnetic resonance imaging mRNA messenger ribonucleic acid MyD88 myeloid differentiation factor 88 NF-κB nuclear factor κB NLR NOD-like receptor NOD1 nucleotide-binding oligomerization domain NOD2 nonobese diabetic (mouse) NOS2A NO synthase 2A nTreg naturally-occurring regulatory T cell PAMP pathogen associated molecular pattern PBMC peripheral blood mononuclear cell PBS phosphate-buffered saline PCR polymerase chain reaction PD-1 programmed death 1 pDC plasmacytoid dendritic cell PFA paraformaldehyde PGN peptidoglycan PI propidium iodide poly I:C polyinosinic:polycytidylic acid; a mimic for double-stranded RNA PRR pattern recognition receptor PSC primary sclerosing cholangitis pSTAT phosphorylated STAT PTPN22 protein tyrosine phosphatase, non-receptor type 22 RANK receptor activator of nuclear factor-κB RIG-I retinoic acid-inducible gene I RLH RIG-I-like helicase RLR RIG-I-like receptor RNA ribonucleic acid RORC2 retinoic acid-related orphan receptor C isoform 2 RORγt retinoic acid-related orphan receptor γt RT-qPCR reverse transcription-quantitative polymerase chain reaction

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RT room temperature RU relative unit SCID severe combined immunodeficiency SES-CD simple endoscopic score for Crohn’s disease SHP-1 src homology 2 domain-containing protein tyrosine phosphatase sib sibling Siglec sialic acid-binding immunoglobulin-like lectin sIL-6R soluble IL-6 receptor SNP single-nucleotide polymorphism SOCS suppressors of cytokine signaling SOD2 superoxide dismutase 2 ssRNA single-stranded RNA STAT signal transducers and activators of transcription T-bet T-box expressed in T cells T1D type 1 diabetes T2D type 2 diabetes Tc cytotoxic T lymphocyte TCR T-cell receptor TGF-β transforming growth factor-β Th T helper TIRAP Toll/interleukin-1 domain-containing adaptor protein TLR toll-like receptor TNBS trinitrobenzene sulfonic acid TNF tumor necrosis factor Tr1 regulatory T cell type 1 TRAIL-DR5 tumor necrosis factor-related apoptosis-inducing ligand-death receptor

5 TRAM TRIF-related adaptor molecule Treg regulatory T cell TRIF TIR (Toll ⁄ IL-1 receptor) domain–containing adapter inducing IFN-β TSLP thymic stromal lymphopoietin TYK2 tyrosine kinase 2 UC ulcerative colitis VIP vasoactive intestinal peptide WT wild type ZnT8A antibodies against zinc transporter 8

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“…try out, and aim at, bold theories, with great informative content; and then let these bold theories compete, by discussing them critically and by testing them severely.”

-Sir Karl R. Popper

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List of original publications

This thesis is based on the following four original publications, which are reprinted with permission of the copyright holders and will be referred to in the text by their Roman numerals:

I. Honkanen J, Nieminen JK, Gao R, Luopajärvi K, Salo HM, Ilonen J, Knip M, Otonkoski T, Vaarala O. IL-17 Immunity in Human Type 1 Diabetes. Journal of Immunology 2010;185:1959-1967.1 Copyright 2010. The American Association of Immunologists, Inc.

II. Nieminen JK, Vakkila J, Salo HM, Ekström N, Härkönen T, Ilonen J, Knip M,Vaarala O. Altered Phenotype of Peripheral Blood Dendritic Cells in PediatricType 1 Diabetes. Diabetes Care 2012;35:2303-2310.Copyright 2012 American Diabetes Association From Diabetes Care®, Vol.35, 2012; 2303-2310 Reprinted with permission from The American DiabetesAssociation.

III. Nieminen JK, Niemi M, Sipponen T, Salo HM, Klemetti P, Färkkilä M,Vakkila J, and Vaarala O. Dendritic Cells from Crohn’s Disease PatientsShow Aberrant STAT1 and STAT3 Signaling. PLOS ONE 2013;8:e70738.

IV. Nieminen JK, Sipponen T, Färkkilä M, and Vaarala O. Monocyte-DerivedDendritic Cells from Crohn’s Disease Patients Exhibit Decreased Ability toActivate Th17 Responses in Memory Cells. Submitted.

1This article appears as an original publication in the thesis Studies of Immune Regulation in Type 1 Diabetes by Jarno Honkanen (2010).

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1 Introduction

In this thesis, properties of human antigen-presenting cells (APCs), particularly those of dendritic cells (DCs), are explored in two immune-mediated disorders, type 1 diabetes (T1D) and Crohn’s disease (CD).

Paul Langerhans observed already in 1868 a cell type in skin samples that is today categorized as a type of DC (1). However, the discovery of DCs is generally considered to date back to the early 1970s, when Ralf M. Steinman and Zanvil A. Cohn described novel murine cells that differed in their morphological characteristics from previously known cell types (2). Later, it was found that DCs potently stimulate the proliferation of lymphocytes in an experimental setting referred to as the mixed leukocyte reaction (3), revealing a distinctive functional feature of these cells. Gradually, the stage was set for a concept of DCs as the initiators and orchestrators of immune responses (4,5). The discovery of DCs was awarded the Nobel Prize in 2011 - dramatically three days after the death of Ralf M. Steinman.

In T1D, the glucose metabolism is severely impaired due to lack of insulin, and the natural course is unfavorable. In 1922 Frederic Banting and Charles Best (6) discovered that affected individuals could be rescued by administering insulin extracts, initially from canine pancreas. Since then, insulin-replacement therapy has formed the basis of the treatment of T1D. Today, T1D is generally considered to result from T-lymphocyte (hereafter referred to as T cell) -mediated autoimmune reactivity against the insulin-producing pancreatic β cells; the disease can be transmitted in animal models by an adoptive transfer of T cells (7) and T cells also make up most of the leukocyte population within the inflamed pancreatic islets in human T1D (8). Typically, diabetes-associated autoantibodies can be found in circulation some years before diagnosis (9,10). However, most individuals that have developed β-cell autoimmunity will not develop diabetes, likely reflecting a scenario in which the autoimmune process is often overcome by tolerogenic mechanisms.

The first description of CD, a chronic inflammatory bowel disease, is often attributed to the Polish surgeon Antoni Lesniowski in 1904 (11). A more profound characterization with 14 patients by Crohn, Ginzburg, and Oppenheimer followed in 1932 (12). Although several treatment regimens are currently available, a cure for CD has remained elusive. It is now widely understood that CD represents an uncontrolled immune reactivity against the generally harmless gut microbiota in genetically prone individuals (13-15), and that an abnormal activation of T cells is likely to play a role in the disease process (16).

T1D and CD share common attributes, most evidently the involvement of the immune mechanisms in the pathogenesis. Moreover, the gut-associated immune system appears to also be relevant in the pathogenesis of T1D (17). However, despite some shared genetic background, no substantial comorbidity exists (18). While the ongoing immune responses towards the innocuous microbiota are clearly a central element in the immunopathogenesis of CD, gut-related microbes may also modulate

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the disease process or even contribute to its initiation in T1D (19,20). In both conditions, a role for aberrant DC function would seem conceivable. We set out to investigate this possibility, aiming also to compare these different types of immune-mediated diseases from the viewpoint of DCs.

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2 Review of the literature 2.1 Dendritic cells DCs represent most specialized types of APCs in the body and have the ability to potently stimulate naive T cells, i.e., T-cell clones that have not yet been activated by their respective cognate antigens (Ags) (21). As will be described in more detail in the chapters that follow, DCs are principally activated by pathogens or other danger signals via germ-line encoded receptors. They then present Ags to T cells via MHC molecules, in order to activate immune responses.

Within the peripheral tissues, a definition which here excludes the primary (thymus) and secondary (lymph nodes, spleen, and the organized mucosa-associated) lymphoid tissues, DCs mostly reside within and beneath the epithelial surfaces, patrolling for microbial invaders (22,23). From the periphery, DCs are generally thought to migrate via the afferent lymphatics to the regional lymph nodes (LNs) (24) where they interact with T cells, activating them (21) (which constitutes the so-called Langerhans-cell paradigm). The migration process is facilitated by changes in chemokine receptor expression pattern, which is related to a process called DC maturation (see chapter 1.3) and is classically due to an encounter with microbial agents or inflammatory cytokines (24). However, there is evidence that at least some types of DCs also migrate to LNs in the absence of inflammation or infection (i.e., the steady-state) (25-27), probably contributing to the maintenance of immunological tolerance (25,28-30). Furthermore, lymphoid-organ resident DCs, such as the well-studied murine splenic cDCs, clearly exist and likely contribute the activated or steady-state presentation of drained soluble Ags in the LN (31).

Despite extensive DC research during the past couple of decades, investigators are only beginning to understand the in vivo precursor-progeny relationships within the DC system (32). In particular, the ontogeny of DC subsets in the peripheral tissues is still somewhat obscure. In mice, both monocytes and committed bone marrow-derived DC precursors contribute to the pool of peripheral tissue DCs (27,29). In addition, it is possible that some DCs, such as skin Langerhans cells, are largely generated from in situ precursors without a constant flux of bone-marrow derived cells (33). Well-established DC-growth factors include Flt3 ligand, GM-CSF, and G-CSF (34-39). The turnover of DCs has been experimentally determined in mouse spleen. After 4 days from the start of bromodeoxyuridine administration, approximately 70% of myeloid and 25% of plasmacytoid DCs (see below) were labeled, indicating longer lifespan for the latter in vivo (40).

Last, it has still been questioned relatively recently whether DCs represent fundamentally distinct populations apart from macrophages, as opposed to a continuum of mononuclear phagocytes with various functional roles (such as the presentation of Ags to naive T cells) (41).

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2.1.1 DC classification and identification DCs can be roughly divided into two major categories: myeloid (mDCs), i.e., conventional dendritic cells (cDCs), and plasmacytoid dendritic cells (pDCs) (42-46). However, cell surface immunophenotyping, functional characteristics, and RNA expression profiling studies suggest that at least five distinct subtypes, the pDCs and four subsets of mDCs, exist in circulation in humans (45,46).

The conventional strategy for the flowcytometric identification or isolation of DCs from the human peripheral blood or from lymphoid tissues, such as that of the tonsils, has been based on the utilization of a mixture of monoclonal antibodies (mAbs) against the lineage (Lin) markers expressed by non-DC cells, usually consisting of anti-CD3, CD14, CD16, CD19, CD20, and CD56 (37,43,47,48). After the exclusion of the lineage-marker positive cells, DCs have typically been identified by their bright HLA-DR expression, and further subdivided by staining against CD11c, and usually also against CD123, utilizing the mutually exclusive high-level expression of these markers by the mDC and pDC populations, respectively (37,47). Additional markers for human blood DCs have been discovered, allowing for simpler identification strategies with more advanced subtyping of mDCs (44).

While there are some shared immunophenotypic markers between the blood-borne DCs and certain cells in the peripheral tissues (23,44,49-53), the relationship between the circulating and tissue DCs is not clear at present (24,49). Nevertheless, based on the in vitro migration characteristics of circulating human DCs (54,55), it seems highly likely that translocation of DCs from blood to the peripheral tissues takes place. In the case of pDCs, the route is probably often different: from the peripheral blood to the secondary lymphoid tissues through specialized structures called high endothelial venules (HEVs) (42,54,56). However, pDCs have also been shown to migrate in the afferent lymph in large mammals (57), contrary to the earlier demonstration of their lack in the intestinal or hepatic lymph in rats (56).

The study of DCs, especially in the human system, is hampered by their scarcity, which has frequently been circumvented by differentiating DCs from monocytes in vitro, most often in the presence of IL-4 and GM-CSF (34). This strategy evidently introduces some difficulties as it is not clear which cell type, if any, these cells would relate to in vivo. The choice of differentiation regimen also influences the properties of the generated DCs (58-60), which, on the other hand, may reflect biologically relevant modulation mechanisms. Some justification for the monocyte-derived DC (moDC) approach is clearly provided by the recent demonstrations that certain DCs in vivo are in fact progeny of monocytes (27,29), although this concept may be contingent on the definition of DC (26).

Another source of difficulty in the identification and categorization of human DC subsets has arisen from the fact that mouse and human DC systems differ considerably in cell-surface immunophenotype (61), and also function. For instance, the expression of the DNA sensor TLR9 is confined to pDCs in human (62), but not in mouse DCs, and the secretion of the cytokine IL-12p70, critical for the priming of

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stable type 1 T-helper (Th) cell clones (63), has been demonstrated from mouse pDCs but not from highly purified human pDCs (64). Thus, a given DC subtype in mice may not accurately reflect the “corresponding” human subtype. 2.1.1.1 Myeloid DCs mDCs are the archetypical class of DCs, being highly efficient in taking up Ags from their environment via endocytosis, presenting them to T cells, and subsequently activating the latter if certain requirements are met (see below) (4,65,66). The range of toll-like receptor (TLR) expression by human mDCs/moDCs spans TLR1-TLR6 and TLR8 (62), through which mDCs can be activated by a wide range of microbial agents. The reported mean or median numbers of circulating mDCs (the CD16+ subpopulation is not considered here; see below) in various studies have typically ranged between 10-25 cells/µl in healthy adults (for examples see: (67,68)), which roughly corresponds to slightly less than 0.3% of leukocytes. The vast majority of human blood mDCs belong to a subpopulation characterized by the expression of CD1c (44). Another, minor subpopulation can be identified by its expression of BDCA3 (CD141) (44) and it typically makes up approximately 10% or less of the total mDCs (69). Knowledge regarding the functional differences between the mDC subtypes is relatively limited. However, several independent studies have demonstrated efficient Ag-cross presentation (i.e., the presentation of exogenous Ags by MHC class I to CD8+ T cells) by BDCA3+ mDCs, suggesting equivalence to mouse CD8α+ DCs (70). On the other hand, the BDCA3-expressing IL-10-induced human moDCs were weak stimulators of allogeneic CD4+ T cells (71), as were the naturally occurring BDCA3+ dermal DCs (by an IL-10-dependent mechanism) (72). The latter also induced CD4+CD25high regulatory T cells (Tregs) with a superior suppressive activity in comparison with CD1c+ DC-induced Tregs.

In addition to CD1c+ and BDCA3+ DCs, human blood contains CD16 (FcRγIII)+, and possibly CD34+ and CD56+ populations with DC-like properties, whose specific functional characteristics as APCs have yet to be fully elucidated. Most DC-identification strategies tend to exclude the relatively frequent CD11c+CD16+CD14dim, otherwise lineage-marker negative cells that have been variably categorized as mDCs, particularly in earlier reports (45,46,73). This population seems to overlap with that designated as CD16+CD14dim monocytes (74), likely depending also on the cut-offs for CD14, CD11c, and HLA-DR. It seems that the allostimulatory function of the CD16+HLA-DR+, otherwise Lin- cells is greater than that of regular CD14+ monocytes (45,75), but their HLA-DR expression has been reported to be lower than that of CD1b/c+ mDCs (45). However, CD16+CD14dim monocytes have also been demonstrated to preferably develop into migratory DCs with an excellent allostimulatory capacity (75), and this subset and its progeny are considered potent APCs (74). Accordingly, categorization of the CD16+HLA-DR+Lin- cells as a type of DCs or monocytes may prove rather arbitrary, given that also regular

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CD14+ monocytes develop into DCs under appropriate conditions. It has been argued that although CD34+ cells can serve as progenitors for DCs, among other types of cells, circulating CD34+HLA-DR+ cells should not themselves be considered as mDCs in the absence of CD86 and CD11c expression (76). In addition, this population is a weaker stimulator of allogeneic MLR than other suggested DC populations and does not seem to upregulate CD83, a marker for mature DCs, in culture (45). Within the CD56+ population in human peripheral blood, a considerable proportion of cells have been reported to spontaneously differentiate into DC-like cells that can be further induced to develop into fully mature CD83+ DCs and can potently stimulate γδ T cells in the CD56+ fraction (77). Of note, as a subpopulation of CD1c+ mDCs also express CD56 (44), it seems possible that some overlap between these subsets exists. 2.1.1.2 Plasmacytoid DCs pDCs constitute a distinct population of DCs in the blood and LNs (42-44,78), and they are also present in the extranodal lymphoid tissues, such as the gut-associated lymphoid tissue (GALT) (79) and that of the tonsils (46,80). In general, pDCs are not easily detected in peripheral tissues in the steady-state (5), but migrate to tissues, such as skin, upon infection, injury or inflammation (81). It has nevertheless been reported in mice that considerable numbers of pDCs also exist in the steady-state within the gut epithelia and lamina propria (LP), especially in the small intestine, lung, and liver (82). In any case, also murine pDCs are recruited to the gut in larger numbers by inflammation (82). Commonly used pDC markers in humans are the interleukin-3 receptor α-chain (CD123), BDCA2 (CD303), and neuropilin-1 (BDCA4, CD304) (44). Among these, BDCA2 is considered a specific marker for pDCs in human peripheral blood (64). Alternatively, pDCs have been identified by their CD4 expression, in combination with lineage-marker and CD11c negativity (83). The reported mean or median numbers of blood pDCs in healthy adults tend to vary between 5-15 cells/µl (likely owing to differences between the various analysis methods; for examples see: (67,68,83)), typical values being roughly equal to just under 0.2% of leukocytes. It has been reported that pDC counts decline with age, in contrast to those of mDCs that appear more or less stable (84).

As early as the late 1970s, studies aimed at the characterization of the type I interferon (IFN)-producing cells in the circulation (85). Only much later, it was discovered (43) that these cells actually represent an already established (47,48) population of DCs, or DC precursors, now commonly known as the pDCs. This emphasizes the dual role of pDCs in the immune system: they rapidly produce high quantities of IFN-α upon activation (86), thus contributing to the first line of innate viral defense, but also develop into potent T-cell activators (47,48,87,88). TLR expression in human pDCs seems to be principally restricted to the intracellular receptors TLR7 and TLR9 that recognize nucleic acids (62,64), which underscores the

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role of pDCs in the induction of antiviral immune responses. pDC activation by bacteria has also been reported (43), but direct activation was not observed in another study using highly purified human pDCs (89). It was originally thought that pDCs have a propensity for type 2 helper T cell (Th2) induction (90), but also Th1-type of CD4+ T-cell activation has since been reported (87,91). However, the generation of sustainable Th1 clones by pDCs is to some extent controversial, as it seems that IFN-α can not substitute for IL-12p70 for the induction of stable expression of the Th1-transcription factor T-bet (63) and IL-12p70 appear not to be readily produced by highly purified human pDCs (86). pDCs have been reported to be capable of some Th17 support in humans (92).

It has been questioned whether pDCs can efficiently present exogenous Ags to T cells, as they lack the mechanisms that allow mDCs to timely enhance the presentation of relevant Ags upon activation (5). In particular, cross-presentation has been absent or weak in murine models (5), although the phenomenon has been demonstrated with human pDCs in vitro (93). Nevertheless, via their role as the principal IFN-α-secreting cells, pDCs may promote cross-presentation by mDCs (94). A recent study also demonstrated that functional pDCs are indeed required for the initiation of robust CD8+ T cell responses in vivo (95). As pDCs express certain receptors that mediate Ag intake, such as BDCA2 and Siglec-H (5), it seems possible that this DC subtype may have a special function in the presentation of some particular types of Ags. This is in contrast to the more universal Ag-intake and presentation machinery of mDCs (5). 2.1.2 DC activation and some elements of its regulation The cardinal feature of the immune system is its ability to distinguish between the self and the non-self, i.e., to identify certain antigenic structures to be associated with potentially hazardous agents, most importantly microbial invaders. In this process, the contribution by DCs is crucial. DCs are equipped with various germline-encoded receptors, many of them highly evolutionarily conserved, which bind to structures abundant in microbes, but not present or scarce in the eukaryotic host (70,96-99). However it is noteworthy that several endogenous ligands capable of some degree of stimulation have been characterized (100,101). In particular, the so-called NOD-like receptors (NLRs, see below) may frequently sense endogenous rather than exogenous factors associated with damage and inflammation (99,102). Actually, an alternative theory for the self vs. non-self concept has been proposed, which claims that the essential quality recognized by the innate immune system, i.e., largely by the DCs, is not “non-self” but “danger” (103).

The microbial structures recognized by the pattern recognition receptors (PRRs) are often referred to as Pathogen Associated Molecular Patterns (PAMPs) (99). These include bacterial peptidoglycan (PGN, a ligand for TLR2) and its derivates γ-d-glutamyl-meso-diaminopimelic acid (NOD1) and muramyl dipeptide

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(=MDP; NOD2), double-stranded RNA (TLR3), lipopolysaccharide (TLR4), flagellin (TLR5), single-stranded RNA (TLR7 and -8), and hypomethylated CpG-rich DNA (TLR9) (15,99). TLR1 and TLR6 are apparently co-receptors that associate with TLR2 as heterodimers, diversifying the range of its ligand specificities to several triacyl and diacyl lipopeptide structures, respectively (99). In addition to TLR heterodimerization, various endogenous accessory molecules have been demonstrated to extend the ligand spectrum of TLRs (104). NOD1 and NOD2, along with several other receptors, belong to the NLR family of chiefly intracellular PRRs. Another class of cytoplasmic PRRs are the cytosolic RIG-I (retinoic acid-inducible gene I) -like receptors (RLRs), i.e., RIG-I-like helicases (RLHs) that include RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) that sense viral RNA (102,105). It seems clear that some sensor molecules remain to be characterized, as responses to pathogen-derived (and incompletely digested self-derived) double-stranded DNA are not satisfactorily explained by the functions of known receptors and pathways (99). It is generally appreciated that cooperation of several PRRs, which is usually the case when naturally-occurring pathogens are encountered, is needed for the initiation of efficient immune responses (98). 2.1.2.1 NF-κB and IRF activation in DCs To translate the event of PRR ligation (i.e., a danger signal) to a meaningful cellular response, APCs utilize a variety of molecules that form complex chains of interacting signaling pathways. For instance, TLRs use four different adaptor proteins alone or in certain combinations for the initiation of the intracellular signaling cascade: MyD88, TRIF, Mal (TIRAP), and TRAM (102,106) (Fig. 1). No TLR- (or IL-1R-, or IL-18R-) mediated signaling can be seen in the absence of MyD88 and TRIF, whereas Mal and TRAM are presumed to act chiefly as accessory adapters for MyD88 and TRIF, respectively (99,102).

Typically, through a series of downstream signaling molecule activations, transcription factors are recruited to initiate the transcription of genes that encode molecules involved in DC activation and maturation (see below). In addition to transcriptional activation, PRR ligation can drive the production of some cytokines, such as IL-1β and IL-18, through the cleavage of preformed precursors. This is carried out via a complex of proteins, which, especially in the case of the NLR family sensor NALP3, is referred to as the inflammasome (102).

A key mediator in DC activation, triggered by the ligation of several different PRRs, as well as IL-1, IL-18, and TNF receptors, is the relatively well-characterized NF-κB pathway (102,107). In immature, quiescent DCs, NF-κB proteins are held in check by a family of inhibitory proteins, inhibitors of κB (IκB) (108). The cascade leading to the activation of NF-κB proteins involves the activation and recruitment of the IκB kinase (IKK) family of molecules, which in turn phosphorylate IκBs to

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license their ubiquitination and degradation (99,102,109). The dissociation of IκBs from the NF-κB proteins exposes the nuclear localization signals of the latter, allowing them to enter the nucleus and initiate the transcription of NF-κB target genes, which include inflammatory cytokines, costimulatory molecules, and chemokines that promote the contact with T-cells (99,108,110,111) (Fig. 1). The resulting upregulation of costimulatory molecules, together with possible upregulation of MHC molecules involved in Ag presentation, is generally referred to as the (phenotypic) DC maturation (65). Besides phosphorylation-induced removal of the regulatory proteins, optimal transcription of several NF-κB target genes also requires phosphorylation of the NF-κB proteins themselves, especially NF-κB p65 (Rel A), which takes place at various residues by multiple kinases (109), contributing to the diversity and fine-tuning of the responses.

A second major class of PRR-induced responses, independent of NF-κB, involves the activation of members of the Interferon Regulatory Factors (IRF) family of molecules, IRF3 and IRF7. The activation of these IRFs leads to production of type I IFNs (a group of cytokines including the various IFN-α subtypes and IFN-β as the key members), which have profound significance in the innate viral defense (99,102,112) (Fig. 1). IRF3 is activated in myeloid APCs in a TRIF-dependent,

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MyD88-independent manner to initiate the production of IFN-β, whereas pDCs use MyD88-dependent activation of IRF7 to induce both IFN-α and IFN-β (112,113) (Fig. 1).

All of the above-described pathways are utilized in TLR signal transduction (99,102). In addition to these key events, several additional kinases and other proteins also participate (102). At least some NLRs, such as NOD2, are capable of direct NF-κB activation, but generally more important might be the IL-1β production via the activation of caspase-1, as in the case of NALP3 (IL-1β in turn activates NF-κB through IL-1R) (102). The LRHs seem to be able to active both NF-κB and the IRFs -3 and -7 (102). As expected, PRR-mediated activation is negatively regulated by various mechanisms, which include alternate splicing of the adaptor proteins, ubiquitinylation/deubiquitinylation, proteolytic cleavage following degradative ubiquitinylation, signal-induced resynthesis of the regulatory proteins, and the relatively recently described microRNAs (99,114). 2.1.2.2 Cytokine-induced signaling and JAK-STAT-pathways in DCs Besides microbial stimuli, DCs are receptive to non-PRR-mediated host signals, such as contact-dependent signaling via cell-surface molecules as exemplified in DC-T-cell interaction (see Chapter 1.3) and cytokines that modulate the activation and maturational status of DCs and, in turn, influence the quality of the adaptive immune responses initiated by DCs. The signals from several cytokines are transmitted from the cell-surface receptors via the JAK-STAT pathways. JAK kinases phosphorylate STAT (Signal Transducers and Activators of Transcription) proteins, and dimerized STATs then function as transcription factors after being transported to the nucleus (115). For instance, cytokines that signal through the STAT1 pathway (IFN-γ, IFN-α) promote (m)DC activation, involving their phenotypic and functional maturation (116), while STAT3-inducing stimuli (IL-10, IL-6) typically restrain DC maturation (80,117,118). JAK-STAT-mediated signaling is subject to negative regulation, most importantly by the Suppressors of Cytokine Signaling (SOCS) family of molecules. Indeed, SCOS action also likely explains certain functional differences between some cytokines with similar STAT activation patterns, as exemplified by the differential role of SOCS3 in the regulation of IL-6 and IL-10 signaling (119). Some cytokines, such as TNF and IL-1β, induce DC maturation largely through the activation of the NF-κB pathway (108), as described above. Furthermore, cytokines have certain roles in the functional programming of DCs, which may be different from their respective actions on T cells. For instance, Th1 cytokine IFN-γ and Th2 cytokine IL-4 both promote Th1-type response induction by upregulating the IL-12p70 production in DCs (120,121), thus also representing examples of positive and negative feedback, respectively (IL-12p70 from DCs and T-cell derived IFN-γ will counteract IL-4 production in T cells).

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2.1.3 Antigen-presentation and T-cell activation by DCs The encounter between DCs and naive T cells is thought to usually take place in the T-cell areas of lymph nodes or other secondary lymphoid organs. This kind of an arrangement ensures that the few T cells with proper T-cell receptor (TCR) specificities will, within the limited amount of time, randomly meet the DCs presenting their cognate Ag.

The general activation requirements of naive T cells include their stimulation through the TCR by the DC-processed peptide bound to the MHC molecules on DCs (i.e., “signal 1.”), and also a costimulatory signal, classically the interaction of DC B7 molecules (CD80 and CD86) and T cell CD28 molecules (i.e., “signal 2.”) (122,123). However, today the picture of T-cell costimulation by DCs is more diversified, with several other costimulatory molecule–ligand pairs contributing to the quality and strength of T-cell activation (124,125). Moreover, some molecules on DCs deliver inhibitory signals for T cells (124) (Fig. 2). In the case of memory T cells, i.e., T cells that have been previously activated or their progeny, the requirements for activation are less strict. Proliferation can be initiated by a weaker TCR stimulus, and effector responses can be carried out in the absence of costimulation (126). Apart from costimulation, some specific molecule pairs mediate adhesion between the DCs and T-cells, thus enabling the interaction (127) (Fig. 2). For CD4+ T helper cells, the recognition of Ag occurs by binding of the TCR to its cognate peptide, i.e., the T cell epitope, bound to the peptide-binding cleft on the MHC class II molecules on DCs or other APCs. CD8+ T cells are instead stimulated by peptides on MHC class I molecules (expressed on most cells in the body). This specificity is governed by selective binding of the CD4 and CD8 to the given MHC molecule (128).

Generally, peptides that derive from material internalized by the DCs will be presented by MHC class II (129). Peptides in the cytosol, which generally are products of cell’s own protein synthesis, are presented by MHC class I (130). However, considerable evidence indicate that DCs are also capable of presenting peptides from externally produced proteins on their MHC class I molecules, a phenomenon referred to as Ag cross-presentation (65). In addition, substantial delivery of peptides from the cytosol to the MHC class II takes place, in which a process called autophagy is likely to play an important role (131). Besides MHC, other molecules also contribute to Ag presentation by DCs, such as the CD1 family of molecules that present lipid Ags.

Aside from cell-contact mediated stimuli, signals from cytokines are needed for optimal T-cell expansion and differentiation. In addition to IL-2, which promotes T-cell survival and clonal expansion in an autocrine fashion, DCs guide T-cell activation towards the appropriate direction by means of cytokine production. A classic example of this is Th1-type of CD4+ T cell differentiation induced by IL-12p70 (132). CD80 and CD86 also seem to exhibit some differences in their influence on T-cell activation, as CD80 has been shown to preferably promote Th1 differentiation, whereas CD86-mediated costimulation enhances Th2 differentiation

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(133). Among DC-secreted cytokines, at least IL-6 promotes Th2-differentiation over Th1 (134). The role of DC-derived cytokines in the differentiation of human Th17 cells is not clear at present, but TGF-β, IL-1β, IL-6, IL-21, and IL-23 have been associated with the differentiation/expansion of the Th17 phenotype (135,136). Notably, the nature of microbial stimuli seems to influence the type of response mounted by the DCs (137). For instance, peptidoglycan and its derivate MDP promote DC-induced Th17 expansion from memory T-cells better than many other PPR-ligands (138). It is also of interest that the anatomical origin/localization of the DCs influences the homing properties of the DC-primed T cells (139). Together, the guidance for appropriate differentiation, mediated largely by the cytokines, is sometimes referred to as “signal 3”. In this manner, DCs act as a bridge between the innate and adaptive immune systems, ensuring that the adaptive system’s potential to match the nearly infinite number of pathogen-associated antigenic structures is accompanied by an appropriate arsenal of effector mechanisms. 2.2 T cells In humans and other jawed vertebrates, the cells of the adaptive immune system consist of B and T lymphocytes. Although both B and T cells originally develop in the bone marrow and are progeny of the same common precursor cell, T-cell progenitors translocate to the thymus for further selection. In the thymus, peptides

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derived from autologous proteins are presented to the developing T cells (140). In contrast to B cells, which can recognize Ags by their cell-surface immunoglobulins in the intact three-dimensional form, T cells and the developing thymocytes rely on MHC-mediated Ag presentation (many T cells with a γδ-type of TCR are an important exception (141)). In order to be functional, thymocytes must bind some of the exact variants of the highly polymorphic MHC molecules carried by that individual (140). Otherwise, they will be deleted in the absence of survival signals (positive selection) (140). If the binding strength between a self-peptide on the MHC and the TCR, on the other hand, exceeds a certain level, the thymocyte will be destroyed as potentially autoreactive (negative selection) (140). This forms the basis for central tolerance. To ensure sufficient diversity in the TCR repertoire, the germ-line encoded TCR genes are rearranged, allowing for a much greater number of different receptor chains (142).

T cells can be classified into αβ- and γδ-cells on the basis of chains present in their TCRs. In contrast to the “regular” αβ-cells, many subsets of γδ-cells have extremely restricted TCR diversities. γδ-cells are rare in the circulation (143), but reside within the epithelia, such as in the skin and the intestine, and actually make up a considerable proportion of the intraepithelial lymphocytes in the latter, particularly in the colon (up to 40% in humans, according to some reports) (144). Most γδ-cells are “double negative”, i.e., they lack the expression of both CD4 and CD8 molecules, although some express CD8 (143). Instead, the vast majority of αβ-cells are positive for either CD4 or CD8. During the thymic residence, αβ-cells are first double negative, then become double positive as the selection proceeds, and finally are left with either CD4 or CD8 molecule (142). This dichotomy has profound consequences, as CD4 and CD8 positive T cells recognize different types of antigenic peptides and exert specialized effector functions. 2.2.1 CD4+ T-helper effector cells For decades, a dichotomous view of T-helper cell polarization prevailed, as it was thought that CD4+ effector cells - the regulatory T cells will be discussed separately below - could essentially be divided into two populations: “Th1” (producing high levels of IFN-γ) and “Th2” (producing mostly IL-4, IL-5, IL-13, and also IL-10) (145). It was not until the beginning of this century that the characterization of the IL-17-producing, IFN-γ- and IL-4-negative cells in mice led to a major revision of this paradigm (146-148). These cells are now commonly referred to as Th17 cells, and more recently Th22 and Th9 subtypes have been identified and named after their respective signature cytokines (149). In addition, follicular helper T cells are generally considered to be a distinct entity.

Th1 cells, largely attributable to their secretion of IFN-γ, enhance pathogen killing by inducing macrophage activation and the production of opsonizing Abs by B cells (145), and they also support CD8 responses (150). Th2 cells assist the

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production of certain types of Abs, particularly of IgE subclass, by inducing a “class shift” to IgE. In addition, they promote the recruitment of eosinophils, which are important to control parasitic infections (145). Th17 cells seem to be of importance for the immune responses against fungi and some bacteria (151). One of their major functions is to attract neutrophils via the IL-17-induced IL-8 secretion from Th17 cells themselves, and also from several other types of cells (152). IL-17 signaling via the promiscuously expressed IL-17 receptor also results in a variable production of other chemokines, (proinflammatory) cytokines and other inflammatory and antimicrobial mediators in a multitude of target cells, including leukocytes, epithelial cells and mesenchymal cells (153). Th17-related cytokines also include IL-17B, -C, -E and -F, whose known functions somewhat differ from those of IL-17A (154), along with IL-22, which is considered to have a protective function in the mucosa (16). IL-27 is thought to be an inhibitor of Th17 cells (151), and IFN-γ and IL-4, the respective Th1 and Th2 signature cytokines, also inhibit Th17 (151).

Th1 cells have been classically considered to account for many autoimmune phenomena, whereas Th2 cells have been implicated in asthma and allergy (148). Since the discovery of Th17 cells, data has been accumulating to suggest that Th17 cells may be responsible for some manifestations of immune pathology (148,155), although their precise contribution in various conditions largely remains to be elucidated. Recently, it has been found that the CD4+ T cell phenotypes are not necessarily stable (149). For instance, cells with a regulatory phenotype (Foxp3+) could be reprogrammed in vivo to become IFN-γ + IL-17A secreting effector cells that cause diabetes upon adoptive transfer (156). Based on the similarities of the suggested requirements (especially that of TGF-β) for the differentiation of the Foxp3+ Treg and Th17 phenotypes (151), this may not be surprising, and it remains to be clarified to what extent this plasticity applies to the different T-helper cell populations, and which mechanisms would drive the phenotypic shifts (149). Importantly, a shift from IL-17 to IFN-γ production may be associated with the acquisition of pathogenic properties in autoimmune and inflammatory diseases (157-159), further underlining the importance of the plasticity concept in immune pathology. 2.2.2 CD8+ cytotoxic T cells The main function of cytotoxic T cells (CTLs) is the killing of infected and transformed cells upon recognition of the MHC class I-bound cognate peptide on the target cell (160). To achieve this, CTLs are equipped with the ability to express various death-promoting effector molecules such as perforin, granzymes, IFN-γ, TNF, and Fas ligand, although the latter may be more important in the control of lymphocyte homeostasis (160,161). The functional specialization of CTLs appears to be less diversified than in CD4+ T helper cells. However, it is noteworthy that CTL division to, e.g., Tc1 (IFN-γ-secreting) and Tc17 (IL-17-secreting) subtypes has been

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proposed, and variable pathogenic potential has been assigned to the latter cell type (162,163). CTLs are of considerable interest in T1D, in which they are widely considered to be involved in the causation of β-cell damage (164,165). 2.2.3 Regulatory T cells The foundations for the modern Treg research were laid by the work of Sakaguchi and colleagues in 1995. This included the description of CD25 as a marker for Tregs able to prevent various autoimmune phenomena – including insulitis - when given to nude mice in conjunction with syngeneic splenic T cells otherwise causing disease (166).

Even earlier, important observations had been made. After the virtual disappearance of the paradigm of suppressor T cells, some of the preliminary findings that fueled the comeback of dominant T-cell-mediated tolerance (which would later take the form of Tregs) were actually related to IBD. Animals receiving transfers of T cells that lacked activation/memory markers developed intestinal inflammation, and the condition was prevented by the administration of T cells expressing certain memory cell markers (167). Furthermore, disruption of the gene encoding IL-2, which is essential for the autocrine proliferation-enhancing loop of effector T cells and the development and especially maintenance of Foxp3+ Tregs (168), was first shown to result in IBD with UC-like features (169). Similarly, the observation of autoimmune-diabetes-inhibiting CD4+ Treg activity in rats dates back to 1993 (170). Another milestone in the modern study of Tregs was undeniably the discovery of Foxp3 as the key transcription factor for the development of the suppressive CD25+ Treg phenotype both in the thymus and in the periphery. Mutations of Foxp3 had been earlier identified as the cause of IPEX-syndrome in humans and the scurfy phenotype in mice (171).

Today, Tregs expressing Foxp3+ are often divided into two categories based on their induction either the thymus (naturally-occurring Tregs, i.e., nTregs) or in the peripheral lymphoid tissues (induced, i.e., iTregs) (172). Several lines of evidence support the idea of functional total affinity, i.e., avidity, of a given thymocyte’s TCRs for MHC class II–bound self peptides as the decisive element for nTreg differentiation in the thymus (168). While negative selection is the fate for thymocytes binding MHC–peptide-complexes with an excessively high affinity, intermediate binding strength, according to this model, favors the Treg pathway of differentiation (168,171).

Although the activation requirements for suppressive CD4+CD25+Foxp3+ Tregs involve antigenic stimulation, activated Foxp3+ Tregs can subsequently exert their suppressive function also in an Ag-nonspecific manner, a principle that applies for the other types of Tregs as well (172-174). Mediators of the suppression by CD4+CD25+Foxp3+ Tregs include immunosuppressive cytokines, such as TGF-β, IL-10, IL-35, and FGL2, molecules related to metabolic regulation, such as cAMP, CD39

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and CD73, cytolytic molecules like granzymes A and B, perforin, TRAIL-DR5, and galectin-1, and membrane-associated molecules that regulate target cells, which include CTLA-4, LAG-3, and Fas ligand (171,172,175). Straightforward competition for IL-2 has also been suggested as a suppressive mechanism of the CD25 (i.e., IL-2Rα) –positive Tregs (176). Notably, much of the suppressive effect of Foxp3+ Tregs is likely mediated via the DCs (172,175). A classic example involves CTLA-4, which, in addition to the theoretically obvious ligand competition (with CD28 for binding to B7), also downregulates DC maturation markers while upregulating the immunosuppressive IDO on DCs (172,175). Furthermore, it is noteworthy that the relationship between the DCs and Tregs is reciprocal in regard to homeostasis. Tregs negatively regulate the numbers of DCs (32), whereas DCs contribute to the maintenance of CD4+CD25+Foxp3+ Treg pool by inducing their polyclonal proliferation (177).

Tr1 cells are a type of CD4+ regulatory cells that lack the expression of either CD25 or Foxp3 (172). They were first described as cells with colitis-preventing potency that produce high levels of IL-10, TGF-β, and IL-5, but only low levels of IL-2 and IFN-γ, and no IL-4 (172,178). Tr1 cells are induced in the presence of IL-10 and mediate their suppressive function largely via production of IL-10 and TGF-β (172,173). These cells play a role in the control of inflammation, e.g., in the intestine, and also in the preservation of self-tolerance (172,173). The nature of Tr1 cells as a distinct entity, instead of a continuum of CD4+ cells with a potential to produce the regulatory IL-10, has been questioned (179). However, it seems conceivable that the net effect of cross-regulation warrants their categorization as a type of Treg and may set them apart from the many types of effector T cells that also produce IL-10.

Th3 cells are TGF-β-secreting CD4+ T cells that were initially characterized as T cells mediating tolerance to orally fed Ag in the experimental autoimmune encephalomyelitis model (180). It is thought that their suppressive effect is mediated via the production of TGF-β, and they seem to be especially involved in the gut-related immunoregulation (172,174). Various subsets of CD8+ T cells with regulatory activities have also been suggested, along with some other populations, e.g., double negative regulatory T cells (172,181). 2.3 Immunological tolerance - with special reference to DCs At the systemic level, T-cell tolerance can be divided to central and peripheral components. The former refers to the thymic screening of developing T cells for their affinity for self-Ags and the resulting deletion of overtly self-reactive cells (described in more detail in chapter 2.2). On the other hand, the latter comprises the many mechanisms utilized in order to limit the activation of cells that have escaped the thymic deletion. DCs, in addition to thymic epithelial cells, have certain roles in the mechanisms of central tolerance; however, these are largely beyond the scope of this

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review. Accordingly, the functions of DCs are next discussed mostly in the context of peripheral tolerance.

At the mechanistic level, the simplest form of DC-mediated T cell tolerance is clearly the hyporesponsiveness or anergy (i.e., state of hyporesponsiveness for future stimulation), which could result when T cells recognize a peptide bound to the MHC, but receive no costimulation (123). This may be the case when DCs have not been sufficiently activated, such as in the absence of pathogen-associated structures or danger signals. Consequently, “immature” DCs would thus be more tolerogenic than their “mature” counterparts due to an insufficient expression of costimulatory molecules. However, the role of DCs is more diversified, as it seems that the induction of active T-cell tolerance (i.e., regulatory properties in T cells) often involves a DC activation of some kind (182), and that T-cell activation can also be actively counteracted by DCs expressing inhibitory molecules (124). It is noteworthy that the activating stimulus for the generation of tolerogenic DCs may not be directly related to the recognition of PAMPs, but may rather be mediated by certain molecules of self origin, such as thymic stromal lymphopoietin (TSLP) for thymic mDCs (183) or Wnt–β-catenin signaling in the periphery (184).

DCs are endowed with the ability to induce various subsets of Tregs. As mentioned above, the naturally occurring CD3+CD25+Foxp3+ Tregs (nTregs) originate in the thymus. mDCs are thought to participate in this process, in which their activation by TSLP seems to be elemental (185). Of note, the phenotype of the TSLP-activated mDCs is highly mature in terms of costimulatory molecule expression (185). pDCs also become responsive to TSLP after activation and promote the development of CD4+CD25+Foxp3+ Tregs when stimulated with TSLP (186). Furthermore, human thymic pDCs induce the development of this Treg subtype from autologous thymocytes when activated by CD40 ligand in the presence of IL-3 (with no TSLP) (187). Hence, by their ability to promote the differentiation of nTregs, DCs also act in the intersection of central and peripheral tolerance.

The specific mechanisms in the induction of CD4+CD25+Foxp3+ iTregs from CD4+CD25-Foxp3- T cells in the peripheral lymphoid tissues may vary depending on the type of DC in question. Nevertheless, it seems clear that TGF-β, either in the milieu or produced by the DCs themselves, has an important role (181,188). In addition, expression IDO, PD-1 ligand 1, and IL-10 (both acting on and secreted by the DCs) have been shown to be involved in this process (181,188). In the context of gut-associated immunity and oral tolerance, retinoic acid and its processing by DCs is of crucial importance, in addition to TGF-β (181,188,189). In skin, on the other hand, RANK-RANK ligand-mediated signaling and vitamin D (1,25-dihydroxyvitamin D3) seem to favor the Foxp3+ Treg inducing function of mDCs (181,188). While thymic induction of nTregs is driven by self-Ag recognition, peripheral iTreg induction is likely to take place upon presentation and recognition of non-self Ags, e.g., those in food and commensal microbiota (171). Of note, TGF-β also acts on DCs, supporting immunological tolerance and normal differentiation of the CD4+CD25+Foxp3+ Treg phenotype (190).

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In mice, Tr1-type of Tregs can be induced by IL-10-producing bone marrow-derived DCs (BMDCs) generated in the presence of exogenous IL-10 (191). Similarly, in the human system, IL-10 production by DCs appears to be required for the Tr1 induction by immature moDCs (192). The so-called “semimaturation” of DCs - characterized by absent/low IL-12p70, IL-6, IL-1β, and TNF production in the presence of costimulatory molecule expression - has also been connected to Tr1 induction (193). Other factors involved in Tr1 promotion by DCs may include IL-27 and the expression of PD-1 ligand 1, HLA-G, and ILT4 on DCs (181), and TGF-β and VIP appear to favor the Tr1/Tr1-like Treg induction by DCs as well (181,194). The role of DCs in the induction of Th3-type of regulatory T cells appears not to be satisfactorily described in the literature, but it would seem logical that TGF-β production by DCs might play a role (174).

Steady-state mDCs (i.e., the pool of mDCs in the absence of any evident maturation stimulus) have been reported to induce CD8+ T cells with a diminished cytotoxic activity and/or tolerogenic properties (195) and also shown to induce Ag-specific anergy in CD4+ T-cells (28). In regard to the induction of proliferation of naturally occurring Ag-specific CD4+CD25+ regulatory cells, mature (CD86high) BMDCs were more effective than the less mature (CD86low) BMDCs (196). On the other hand, several studies have demonstrated that high-level costimulation does not favor the peripheral de novo induction of Foxp3+ Tregs (171). Conversely, TCR-ligation of proper strength plus “suboptimal” costimulation promotes iTreg differentiation (171), which seems logical from the viewpoint of limiting extensive reactivity against harmless non-self Ags. Furthermore, this largely accounts for the rationale in attempts to induce systemic tolerance by a continuous administration of small doses of relevant Ag – often successful in experimental models (171). However, at the mechanistic level there is considerable overlap between the mechanisms that induce active tolerance and those that induce mere hyporesponsiveness/anergy, as demonstrated, e.g., for ICOS – ICOS ligand-mediated signaling (197). Furthermore, when Ag is introduced the context of dying cells, DC-mediated Ag-specific T-cell tolerance results (198), which obviously helps to preserve tolerance to self in the periphery.

During the past decade, a considerable body of evidence has accumulated to suggest that pDCs may play a key role in the induction and maintenance of immunologic tolerance by several mechanisms, although many of these are clearly not exclusive to this DC subtype. pDCs mediate tolerance to orally fed Ags, i.e., oral tolerance (199), and may also exert a tolerogenic function in several other contexts such as transplantation (200) and autoimmunity (201). Human pDCs can prime regulatory T cells with a non-Ag-specific suppressive potential (202), induce anergy in Ag-specific CD4+ T-cell lines (203), and promote the generation of IL-10-secreting CD4+, i.e., Tr1-type of cells (88) and CD8+ T cells (204) with a regulatory capacity. The priming of Th1-like responses by pDCs also seems to be accompanied by a considerable IL-10 induction in T cells (87). Facilitating their regulatory activities on

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T-cell function, pDCs express certain well-characterized tolerance-promoting molecules, such as IDO and PD-1 ligand 1 (205).

Yet another tolerogenic mechanism of DCs could be a straightforward deletion of self-reactive T cells in the periphery (123,195,206), as in the negative selection process in central tolerance. AIRE-positivity has also been demonstrated in human peripheral lymphoid tissue cells positive for DC-markers. These cells expressed tissue-restricted Ags and were positive for IDO and IL-10 (207). Huge overall significance for DCs in the maintenance of tolerance is suggested by the report of fatal autoimmunity by a constitutive DC ablation in a genetic model (208), but it has been argued that the condition may have actually represented a myeloproliferative syndrome caused by the systemically elevated Flt3 ligand levels in the absence of DCs (209). 2.4 Some aspects of DCs in gut-related immunoregulation From an immunological viewpoint, the alimentary tract represents an interface between the body and the environment, similar to the skin and the respiratory epithelium. The specific challenge is to tolerate Ags associated with beneficial factors, such as food and commensal microbiota, while robustly and discretely expelling the undesired pathogens. Failure to do so may result in gut inflammation, such as in CD, or possibly in autoimmunity, as in T1D; the latter condition is also connected to gut-associated immunity, including intestinal DCs, by several lines of evidence (17,210). Immune cells in the intestine, including DCs, are scattered along the LP, reside within the epithelia, and populate Peyer’s patches, which are structures specialized in Ag sampling and presentation (22,211). By default, many features in gut-associated immunity are tuned for the induction of tolerance. For instance, tolerogenic DCs, and subsequently regulatory T cells (Foxp3+ iTregs, Tr1/Tr1-like, and Th3 cells), are readily induced in the GALT and/or mesenterial LNs (MLNs) under the influence of substances rich in the normal intestinal environment, such as vitamin A and VIP (30,171,194). Furthermore, gut DCs show reduced reactivity when stimulated with endotoxin (LPS), a major constituent of the cell wall in gram-negative bacteria (212).

Based on animal studies, DC populations in the gut are thought to derive both from monocytes and from dedicated DC precursors. CD103+CX3CR1- DCs in the LP, which in mice represent the majority of CD11c+CX3CR1- DCs (211), are progeny of dedicated precursors (pre-cDCs), induce preferably Foxp3+ Tregs in a TGF-β- and retinoic acid-dependent manner, and function as migratory DCs that translocate to the MLNs also in the steady-state (27,29,189,213). For Foxp3+ Treg induction, CD103+ DCs are endowed with an enhanced capacity to metabolize vitamin A to generate retinoic acid (214). Furthermore, the expression of the immunoregulatory IDO is also highest in the CD103+ DC fraction (215). Wnt-Beta-catenin-signaling in DCs is critically involved in the DC-mediated induction of Foxp3+ Tregs and tolerance in the

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gut and may actually be required for the aforementioned activities by retinoic acid and TGF-β (184).

CD103-CX3CR1+ DCs, on the other hand, are of monocyte origin and sample Ags by extending their processes to the gut lumen (27,29,216). It has been suggested that CX3CR1+ cells do not migrate in the lymph, are inefficient to prime naive T cells, and generally do not express high levels of CD11c (26,27). Because of these features, it has been argued that the CX3CR1+ cells should not be considered classical CD11c+ mDCs (26), although the opposite has recently been demonstrated for CD103-CX3CR1int DCs when gated based on high-level positivity for CD11c (217). As a whole, the reported phenotypic diversity within the CD11c+ mononuclear cells from mouse intestinal LP is considerable, and classification of every subset as a type of DC or macrophage, let alone its necessity, is not presently clear (211). CD103-

/predominantly CD103-,CD11b+ DCs, with high CD11c expression, from LP or intestinal lymph have been assigned with the propensity for the induction of IL-17 in T cells (217,218). Also in humans, CD103+ DCs are found both in the small intestine LP and MLNs (30,219), but most aspects regarding the equivalence between the mouse and human intestinal DCs remain unresolved.

In addition to mDC populations, pDCs are also found in mouse GALT (79,82), and, as mentioned above, are increased by inflammation (82). In humans, pDC counts in the colonic LP and MLNs have been reported to be increased by the IBD-associated inflammation (220), although not consistently (39,49,221). In another relatively recent study, 0.5–1% of isolated LP mononuclear cells from non-IBD human colon and jejunum showed a phenotype compatible with mature CD1c+ mDCs, while the number of cells expressing the pDC marker BDCA2 was low (53). However, some caution is warranted in the interpretation, as BDCA2 is known to be downregulated upon pDC maturation (44). Other researchers have found only low numbers of both CD1c+ and BDCA2+ cells in the colon in situ, whereas the number of BDCA3+ cells was relatively high in IBD and non-IBD colon and MLNs (49). 2.5 Type 1 diabetes Type 1 diabetes (T1D) ultimately results from the destruction of the insulin-producing β cells in the pancreatic islets. Substantial comorbidity accompanies T1D in the form of macrovascular complications, which include coronary artery disease, peripheral arterial disease, and cerebrovascular disease, and also as microvascular complications, i.e., nephropathy, retinopathy, and neuropathy (222).

In Finland, the incidence of T1D is among the highest in the world with more than 60 per 100 000 children below the age of 15 years affected (223-225). High incidence rates are also seen in other wealthy western nations (223-225). The incidence in the industrialized countries has been increasing considerably since the mid-nineteenth century (223,225,226). During the last couple of decades, this trend has been increasing especially in the young children with less than five years of age

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(223). However, this development may now have started to show signs of leveling off, as reported from Sweden (226).

Overt T1D occurs when the β-cell mass falls to the level no longer able to produce an adequate supply of insulin and thereby looses blood-glucose control. However, the process of β-cell destruction generally starts years before, as indicated by the emergence of diabetes-associated autoantibodies (AAbs) in the circulation (9,10). Given that the detection of AAbs is often not followed by actual disease (10), this interval, referred to as the prediabetes, may offer enormous possibility for preventive measures. 2.5.1 Diagnosis and classification of diabetes According to WHO criteria, diabetes is diagnosed when the level of glucose in the plasma is 7.0 mmol/l or higher at fasting, or exceeds 11.0 mmol/L after two hours of oral administration of 75 g of glucose. A milder deviation from normal values (which are <6.1 and <7.8 mmol/l, respectively) represents either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). These conditions indicate increased risk for developing diabetes (224). By WHO criteria, diagnosis may also be made based on elevated levels of glycosylated hemoglobin (HbA1c).

No definitive globally used criteria exists today for the differential diagnosis between T1D and other forms of diabetes (224,227). The diagnosis of T1D is established by a combination of clinical and laboratory criteria/parameters, including typical symptoms, age at the onset, body weight, insulin dependency, presence of diabetic ketoacidosis or elevated levels of ketone bodies in the blood/urine, C-peptide measurement, and possibly the assessment of some diabetes-associated AAbs (224,227), recommended by some authors routinely in the diagnosis of T1D (227). Diabetes-associated AAbs include islet cell AAbs (ICA, generally determined by indirect immunofluorescence on human pancreas), and the biochemically defined Abs against insulin (IAA), glutamate decarboxylase (GADA), islet antigen 2 (IA-2A), and zinc transporter 8 (ZnT8A) (228).

Although autoimmune diabetes is usually diagnosed in childhood, adolescence, or early adulthood, insulin-dependent diabetes with autoimmune features is also often encountered later in life (224,227). Autoimmune diabetes in the adulthood (commonly at the age of 30 or older) frequently presents without the requirement of insulin at diagnosis, and is then considered to be Latent Autoimmune Diabetes in Adults (LADA) (229). LADA can be distinguished from type 2 diabetes (T2D) by the presence of GADA and by a combination of clinical features (such as lack of obesity and the presence of classical symptoms of diabetes, such as polyuria, polydipsia, and possibly unintentional loss of weight) (229). LADA patients will often develop insulin-dependent diabetes during a follow-up of a few years (229). Some classifications of diabetes have T1bD as an idiopathic category (224,227) that includes the relatively recently described fulminant type 1 diabetes with a suspected

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viral etiology and especially found in East Asia, and the ketosis-prone subtype encountered in sub-Saharan Africa and among the U.S. African-American population (227). Maturity-Onset Diabetes of the Young (MODY), or monogenic diabetes, is a heterogeneous group of genetic disorders with an autosomal dominant inheritance affecting β-cell development or the regulation of β-cell function (230). The onset is typically before the age of 25 years (230). MODY patients may develop AAbs (224,227). Neonatal diabetes is a rare disorder that generally manifests itself within the first six months of life, lacks signs of autoimmunity, and can be either transient or permanent (231). The former is often a consequence of abnormal imprinting of certain areas in the genome, whereas the latter results from a variety of severe monogenic defects in genes regulating the pancreatic development or function, including mutations in the insulin gene itself (231).

T2D typically manifests at older age, is often accompanied by insulin resistance, is related to a wider range of metabolic disturbances (232), and lacks signs of autoimmune phenomena (224). Pregnancy is associated with an elevated incidence of diabetes, and this condition, referred to as gestational diabetes, is related to an increased risk of developing diabetes later in life, most commonly T2D. Disease conditions affecting the endocrine pancreas in a non-specific manner, such as pancreatitis, and/or removal/resection of the pancreas will also lead to an insulin-dependent diabetes if the loss of functional endocrine tissue is sufficient (secondary diabetes). 2.5.2 Pathogenesis of T1D It is indisputable that T1D is a product of genetic predisposition and environmental factors (233). Genetics play a central role in the susceptibility for T1D: the reported probandwise concordance rates have been around 40% or higher in homozygotic twins, compared to those around 10% in dizygotic twins (234). At least 40% of the familial clustering is attributed to HLA (235). The strongest genetic risk determinant is the HLA-DQB1 locus within the HLA class II region (which contains the genes encoding MHC class II molecules), but the risk is modified by other loci as well (235). Particular high-risk HLA genotypes are associated with certain patterns of AAb development (236), which underscores the role of HLA class II-mediated Ag-presentation. It is of interest that the HLA-DQB1 locus may also confer strong dominant protection against T1D, most importantly by the DQB1*0602 allele (235). Given that many other T1D susceptibility genes encode molecules that are involved in the activation and regulation of the immune system, e.g., PTPN22, IL-2RA, and CTLA-4 (237), genetics constitute an important contribution to the notion of T1D as an immune-mediated disease, further supported by the involvement of diabetes in the monogenic autoimmune polyendocrine syndromes APECED and IPEX (227). The plethora of T1D susceptibility alleles associated with immunoregulation - most of

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them with relatively low odds ratios - also imply a variety of subtle mechanisms with disastrous consequences when the resulting autoimmunity is directed towards the estimated 1g of human β-cell mass. In contrast to the relatively well-defined genetics of T1D, environmental factors in the disease process have, despite considerable efforts, remained insufficiently characterized, and no compelling evidence for an environmental cause exists to date. Among suggested triggers/sustaining factors of β-cell autoimmunity are microbes, especially enteroviruses (20), exposure to certain toxic agents such as N-nitroso compounds, (238), intake of nutritional factors like vitamin D (239) – which among its other functions is a DC modulator - and early exposure to cow´s milk in the form of cow’s milk-based formula (240). It has been further proposed that bovine insulin, present in cow’s milk and showing a strong sequence homology to human insulin, could serve as the autoimmunity-initiating Ag (17). This is compatible with the observation that IAA are commonly the first class of AAbs to appear (228), but the primacy of insulin as an T1D auto-Ag may also be readily explained by other mechanisms, as exemplified by the connection between diabetes development and altered or insufficient thymic presentation of insulin (233). Nonetheless, children progressing to diabetes often develop AAbs before the age of two years (9,10), supporting the idea of disease-initiating environmental factors acting early in life. The association of microbes to the disease process, seen both in human studies and in experimental models (19,20,241), comes with a variety of possible links to β-cell autoimmunity and destruction, but also to disease prevention. Supported by experimental and/or descriptive evidence are the infection of β-cells, activation of innate immunity (which may be associated with both disease promotion and prevention), by-stander activation of the immune system via tissue destruction/phenotypic changes of non-immune cells and the associated release of auto-Ags, and mechanisms of molecular mimicry (241). Microbes and microbial infections are also intimately connected to the hygiene hypothesis, which has during the last decades extended from allergy to autoimmune diseases, such as T1D. Generally speaking, bacteria and parasites usually play a protective role in the animal models of T1D, while the role of viruses may either be disease-promoting or protective (241).

In light of these general observations, altered DC and T-cell functions could contribute to the disease process by several mechanisms. These may include enhanced induction and/or impaired regulation of self-reactive effector T cells, insufficient activation of antimicrobial effector T cells, and attenuated generation and/or function of protective Tregs (see below). Despite the well-established role of diabetes-associated AAbs in the detection of β-cell autoimmunity, humoral immunity has not been considered to play a substantial role in the pathogenesis (233). However, there is evidence that B cells may promote disease development by other mechanisms, including their function as APCs for diabetogenic T cells, as suggested by the requirement of B-cell-bound Ig in the disease process (242) in the NOD model (see below). Therapeutic targeting of B cells via CD20 has shown some beneficial effects

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both in animal models and human disease, although regulatory functions by B cells have also been reported (165,233,241). NK-cells are present in the infiltrates in the islets in animal models (also prior to T cells) and in human T1D, and their numbers or activation has been reported to correlate with the destructiveness of the infiltrate. Several lines of mechanistic evidence demonstrate their pathogenicity in animal models, but also protective activities have been described (241).

Finally, it is currently not clear which immune-cell type, if any, would be the most crucial one in the initiation of the disease process, and, at least in mice, the initial release of β-cell Ags occurs because of normal turnover of β cells (233,243). Given this physiological availability of β-cell Ags, it is of considerable interest whether an altered activation state or migration of DCs contributes to the disease initiation. 2.5.2.1 T cells in T1D Autoimmune diabetes can be transferred in mice by a transfer of T cells, providing a clear indication of their role in the disease process (7). Both CD4+ an CD8+ populations seem to play a role based on the cell transfer experiments. Much of the experimental work related to the role of T cells, and to many other research questions in T1D, has been carried out in the two principal genetic models of autoimmune diabetes, the nonobese diabetic (NOD) mouse and the Biobreeding (BB) rat (233,241,242,244). In the NOD model and also in recent-onset human T1D, CD4+ and CD8+ cells are found within the inflamed islets. A predominance of CD4+ cells exists the former (244), whereas in human subjects more CD8+ cells are seen (8). CD8+ cells in the human T1D islets have recently been shown to be indeed islet-autoreactive, with Ag-specificities overlapping with known B-cell Ags, including insulin (and preproinsulin), IA-2, and GAD (164).

Traditionally, T1D has been considered a Th1-mediated disease (241), which fits to the observations that Th1 cells can transfer disease (233), and that IFN-γ can mediate β-cell destruction by inducing reactive-oxygen species (241). However, IFN-γ-deficient NOD mice are not protected, and T-bet deficiency actually ameliorates disease (233). In the NOD model, interventions that promote a shift from Th1 to Th2 responses – some of them utilizing a transfer of DCs – have been associated with disease protection in the NOD model (165), but this may, at least occasionally, reflect a secondary effect more than the cause of the disease resistance (165).

Relatively recently, an elevated frequency of IL-17-producing cells was reported in anti-CD3-stimulated PBMC cultures from long-term T1D patients, but not in recent-onset disease (245). In a study by Jain and colleagues in mice, Th17-polarized transgenic BDC2.5 T cells (recognizing β-cell auto-Ag in the NOD mice) caused disease when transferred to NOD-SCID recipients; this was inhibited by anti-IL-17 mAb (246). In the context of spontaneously developing disease in NOD mice, IL-17 inhibition prevented disease only if given to animals with 10 weeks of age, but

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not in younger mice (247). On the other hand, the transfer of in vivo differentiated BDC2.5 Th17 cells from NOD to NOD-SCID mice promoted disease just as the transfer of Th1 cells did, but this was associated with a phenotype shift to Th1 (157). In another study with similar results, anti-IL-17-treatment reduced the severity of insulitis, reinforcing evidence for an accessory role for Th17 cells in the BDC2.5 - NOD-SCID model (158). However, in the study by Jain et al., neutralization of IFN-γ actually led to a reciprocal increase in Th17 cells and reversal of the protective effect in the context of (tolerance promoting) GAD-Ig-construct administration (246). Given these discrepant findings, it appears that the role of Th17 cells and IL-17 is not clear in the NOD model.

There is considerable evidence that Tregs may counteract the disease process. First, IPEX patients with mutated FOXP3 gene develop autoimmune diabetes early in life, as do scurfy mice with the equivalent genetic defect (171). Second, disease in the NOD model can be inhibited by inducing Foxp3+ Tregs using a short pulse of TGF-β in the islets (248). Third, the depletion of Tregs in the BCD2.5-NOD model rapidly causes activation of islet infiltrating NK cells and an overt disease (249). Finally, disease progression in the NOD mice is associated with a decreasing Treg/T-effector cell ratio in the inflamed islets (250), and “ex-Tregs” (of both nTreg and iTreg origin) that have lost their Foxp3 expression start to produce IFN-γ and IL-17, accumulate in the islets of NOD mice, and cause disease when transferred to NOD x Tcra-/- mice (156). It has also been suggested that the mechanism of the anti-CD3-treatment known to prevent or even reverse disease in the NOD model and preserve β-cell function in human T1D likely involves a specific preservation of Tregs (233). Contradictory reports exist regarding numerical defects of Foxp3+ Tregs in human T1D (251-253). Recently, a decreased percentage of CD4+CD25+Foxp3+ Tregs was detected among CD3+ cells in duodenal biopsies from T1D patients. This was accompanied with a reduced capacity of in vitro-differentiated CD11c+CD103+ cells from the biopsies to convert CD4+ effector T cells into Foxp3+ Tregs (210). Attenuated suppressive potential of Tregs has been described as well (254), but not consistently (252). However, also in human recent-onset T1D, an elevated proportion of Foxp3+ cells has been reported to produce IFN-γ, possibly indicative of greater plasticity of the Treg phenotype in patients (255). 2.5.2.2 DCs and monocyte/macrophages in autoimmune diabetes – lessons from animal models In the NOD model it has been demonstrated that CD11c+ mDCs are the first immune cell type to appear in the pancreatic islets, collect islet-cell Ags released by β-cell turnover, and migrate to pancreatic LNs where they prime diabetogenic T cells (243). There is evidence that the genetic background in the NOD model leads to an altered function of mDCs, such as an enhanced activity of the NF-κB pathway, IL-12p70 and TNF production, and B7 expression (256-258). The increased NF-κB activation has

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been demonstrated to account for the enhanced immunostimulatory activity seen in DCs from NOD mice, including the secretion of IL-12p70 (256). However, also reduced IL-12p70 production by NOD BMDCs has been reported (259). Disease was nevertheless inhibited in the NOD model by the administration of DCs deficient in NF-κB activity (260) or by inhibiting costimulation with antisense oligonucleotides, which was associated with an increase in the number of CD4+CD25+ putative Tregs in spleen (261). On the other hand, MyD88-deficient NOD mice developed diabetes when housed in germ-free environment but were protected in specific pathogen-free environment, via a mechanism possibly related to changes in gut microbiota due to MyD88 deficiency. Furthermore, the disease was attenuated in germ-free MyD88-deficient NOD mice when colonized with bacterial species common to normal human flora (19).

Although an enhanced generation of DCs has also been described in NOD mice (257), it seems relatively well known that these mice, as well as the BB rat, have some developmental/maturational deficiencies in their monocyte/DC/macrophage lineages (36,262-264). Furthermore, experimental procedures aimed at correcting some deficiencies/imbalances have proven efficient in inhibiting diabetes, such as the administration of Flt3-ligand (35,36).

Several groups have found that type I IFN may promote diabetes development in mouse models, including the NOD model (241), which alone suggests a role for pDCs, the principal IFN-α-secreting cells. On the other hand, many aspects of pDC function are in favor for their function as tolerance-inducing/preserving DCs, as outlined in chapter 2.3. Direct evidence connecting pDCs to the protection from autoimmune diabetes include the demonstration of disease inhibition via CD4+ T cell regulation and the promotion of IDO production in the islets, in a setting where mDCs aggravated disease (265). In another report, preventive effect of G-CSF administration in the NOD model was associated with the recruitment of pDCs and CD4+CD25+ Tregs (38). However, as pDC depletion has also been demonstrated to delay disease in NOD mice (266), results are contradictory.

Macrophages (which, like some DCs, are progeny of monocytes) are readily found within the islets in the NOD model – also prior to the appearance of T cells – and also in human cadaver T1D pancreata (8,241). Possible mechanisms by which macrophages may contribute to the destruction of β cells likely involve their production of IL-1β, TNF, and reactive oxygen species (241). NF-κB hyperactivation has also been observed in macrophages of NOD mice, which resulted in an enhanced production of IL-12p70, TNF, and IL-1α, but not in elevated T-cell stimulatory activity (267). 2.5.2.3 DCs and monocyte/macrophages in human T1D There are several papers on the numbers and some other characteristics of circulating DCs in T1D patients, with highly inconsistent findings. In a brief report by Peng and

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colleagues in 2003 (268), increased total DC numbers were found in patients and in individuals at risk, with a reduced mDC/pDC ratio. Increased HLA-DR expression was also reported, and PBMCs from the patients and in-risk subjects exhibited elevated spontaneous IFN-α production, which was mostly attributed to pDCs. Summers et al. (269) found no differences in the relative or absolute DC counts or in the mDC/pDC ratio. In this study, IFN-α production was reduced in the enriched DC fractions from patients when stimulated with CD40 ligand +/- IFN-γ. No phenotypic alterations were observed, and, with the limited number of individuals tested, no differences in the DC-induced T-cell proliferation were seen. Study subjects were both children and adults. Vuckovic and co-authors (270) studied children with a recent-onset disease, and children and adolescence with an established disease, with an appropriately age-matched control group, and found decreased relative and absolute counts of both mDCs and pDCs. Unusually high mDC counts were reported, which may be due to the inclusion of the CD11c+CD16+ subpopulation (see (73)), although this was not explicitly disclosed. No phenotypic differences were detected. In contrast to some earlier findings, Chen et al. (271) reported reduced pDC counts and an increased mDC/pDC ratio, but the age of the study subjects and disease duration varied considerably. Allen and colleagues (272) found comparable relative DC counts in recent-onset patients and controls, but within the total DC fraction pDCs were proportionally increased in T1D. Authors also demonstrated enhanced IFN-γ production by an influenza specific CD4+ T cell clone when Ag (IA-2 modified to contain influenza hemagglutinin CD4+ T-cell epitope) was presented by pDCs in the presence of IA-2A+ serum, indicating synergy between the pDCs and AAbs in the activation of autoreactive T cells. Finally, Meyers and co-authors (273) observed altered TLR signaling in patients, including an increased IFN-α production by resiquimod (TLR7/8 ligand) -stimulated pDCs. Furthermore, LPS-induced phosphorylation of NF-κB p65 was enhanced in mDCs and monocytes from patients. Circulating IFN-α2, IL-1β, IFN-γ, and CXCL-10 were also found to be increased in T1D in multiple-analyte testing. Study subjects were both children and adults diagnosed within six months, with suboptimal age matching. No alterations of DC numbers were found in this study.

Several groups have also studied moDCs in human T1D. In 1995, it was found that the generation of DCs from the monocyte precursors was abnormally weak in patients, with a reduced potency to stimulate allogeneic and autologous T cells in MLR (274). Similar findings, extending to poor phenotypic maturation as assessed by B7 upregulation, have later been reported both in recent-onset patients and in individuals at risk (275-278). Also in moDCs, aberrant LPS-induced activation of the NF-κB pathway has been observed in T1D (279), and some authors have found increased IL-6 and/or IL-10 production (276,278,280) and decreased secretion of IL-12p70 (278). In a recent report, monocytes from T1D patients spontaneously produced increased amounts of IL-1β and IL-6, which was suggested to promote the enhanced induction of IL-17-producing cells the authors demonstrated in T1D (245). Macrophages (CD68+) are found together with DCs (CD11c+) in the islets of recent-

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onset T1D patients, where both subsets showed signs of TNF and IL-1β production (281).

2.6 Crohn’s disease Crohn’s disease (CD) represents one of the two major forms of IBD, the other being ulcerative colitis (UC). Like that of T1D, the incidence of CD has been increasing in the industrialized western nations since the mid-nineteenth century (282), being in its last one or two decades in the range of 4-10:100 000 in the Western Europe (282). Many aspects related to the urbanization have been implicated in the increasing incidence, but currently smoking remains the best studied and the sole well-established predisposing environmental factor (13). The onset may be either in the childhood or later in life, but there is an incidence peak in the early adulthood (13). CD can affect the gastrointestinal tract in its entirety, setting it apart from UC. Extraintestinal manifestations of CD may affect liver, eyes, skin and joints, specific entities consisting of primary sclerosing cholangitis (PSC), peripheral and axial (i.e., sacroileitis and ankylosing spondylitis) arthritis, episcleritis, uveitis, erythema nodosum and pyoderma gangrenosum. The risk for small bowel and colorectal cancer is increased in CD patients (13), and the presence of PSC, although more frequent in UC than in CD, is a determinant of IBD-related cancer risk via an increased susceptibility for cholangiocarsinoma and colorectal cancer. CD is associated with moderately increased overall mortality, with reported standardized mortality ratios of approximately 1.5 fold (283). 2.6.1 Diagnosis and treatment of CD The diagnosis of CD is clinical and based on patient history, physical findings, endoscopy (primarily ileocolonoscopy with biopsies), data from imaging and laboratory studies, and histopathology (13,284). Symptoms of CD include diarrhea, abdominal pain, rectal bleeding or mucus in the stool, fever, and weight loss (13). The disease may also present with clinical signs of bowel obstruction (13). CT or MRI enterography, and possibly capsule endoscopy are used as imaging modalities (13,284). Endoscopic characteristics peculiar to CD are ileal inflammation, discontinuous involvement, cobblestoning, fissural and longitudinal ulcers, strictures, and fistulas (13,284). Histologically, CD-associated features include focal chronic (lymphocytic) inflammation and patchy chronic inflammation, focal crypt irregularity and granulomas, and in the ileum also an irregular villous architecture. Transmucosal inflammation is characteristic of CD (284). In addition to standard laboratory studies, newer biomarkers such as fecal calprotectin have been adopted for clinical use (13). After diagnosis, CD is generally classified according to the Montreal classification,

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which involves categories for the age at diagnosis, localization, and behavior (the latter will typically vary during follow-up) (13).

Commonly used drugs to treat CD include aminosalicylates (mesalazine (=5-ASA) and sulfasalazine), corticosteroids, antimetabolites (azathioprine, 6-mercaptopurine, methotrexate), and the newer biological agents, such as TNF inhibitors (infliximab, adalimumab, certolizumab) and the α4-integrin antagonist natalizumab (13,285). Antibiotics, such as ciprofloxacin and metronidazol, are also used, but in a recent systematic review were deemed unlikely to induce remission, although symptoms may be improved (285). Numerous novel biological agents, including inhibitors of cytokines other than TNF, are currently been tested for IBD (286). Surgical operations (strictureplasty, bowel resection) are needed in most patients at some point. 2.6.2 Pathogenesis of CD As in T1D, twin studies have demonstrated a clear genetic component in CD, with probandwise concordance rates in the range of 50-60% for monozygotic and <7% for dizygotic twins (287). Contribution of the genome-wide association studies (GWASs) to the hypothesis formation and functional understanding of the disease process has been enormous. For instance, the likely involvement of the autophagy mechanisms was discovered largely owing to GWASs, and IL-23R, in addition to many other IL-23/Th17 pathway-related (TYK2, JAK2, STAT3, CCR6 and ICOS ligand) and other immune-related loci have been revealed in this way (288). To put things into perspective, the missing genetic contribution to disease susceptibility was in 2010 calculated at 77%, however (with 71 confirmed loci) (18).

It is likely that CD represents a failure in the mutualism between the human host and the intestinal commensal flora; the gut microbiota, which is tolerated in healthy subjects, appears to drive inflammatory immune responses in the affected individuals (13-15). Based both on GWASs and animal studies, it seems that the symbiosis may be disrupted at several levels, starting from the physical factors that maintain the intestinal barrier integrity, such as mucus production and tight junctions between the intestinal epithelial cells (IECs) (13).

Some of the various levels at which genetic defects may exert their effects are likely mirrored in the known functions of NOD2. SNPs in the NOD2 gene are the strongest genetic risk-determinants for CD, with well-documented odds ratios of approximately 2.5 for simple heterozygotes and 17 for homozygotes and compound heterozygotes (14). These variants impact epithelial and phagocytic cells causing deficiencies in epithelial-barrier function (289). Another likely influence of the mutated NOD2 is a defective induction of autophagy, e.g., in DCs, (290), a phenomenon implicated by other CD-susceptibility loci and functional studies as well (13). In DCs, the CD-associated NOD2 variants also cause hyporesponsive cytokine response upon activation with MDP, a derivate of bacterial PGN (138). In addition,

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NOD2 mutations have been connected to an impaired Paneth cell α-defensin production, which has direct consequences for the first-line bacterial defense (289).

Specific infectious etiology has also been proposed, most frequently infection by Mycobacterium avium subsp. paratuberculosis, but no convincing evidence for such association, let alone causal role, exists to date. However, it is noteworthy that genome-wide association studies implicate mechanisms relevant in anti-mycobacterial defense, including shared susceptibility loci with leprosy and those involved in autophagy (13), and that intestinal tuberculosis may also closely resemble CD.

Although a single causative microbial agent could not have been identified, it is well-established that patients with CD have profound alterations in the their intestinal flora, which consists of both altered composition of species and reduced diversity (291). Many aspects of modern urban life may alter the gut microbiota in comparison with rural lifestyle, and some of them, such as diet, are also among the suspected environmental factors in CD (291). However, it is not currently known whether these alterations represent a causative factor or a secondary phenomenon. A plausible answer could be both, given the aforementioned evidence strongly implicating defective first-line bacterial defense. Certain species may invade the niche created by permissive changes in the innate immune reactivity/non-immune mechanisms within the epithelia, outgrowing some other. The disturbed balance might then lead to an altered crosstalk between the microbiota and host (291-293) and cause disease that is ultimately driven by aberrant effector responses, likely accompanied by a defective immune regulation towards a multitude of Ags (see chapters 2.3 and 2.4). 2.6.2.1 CD and STAT3 The association of STAT3 with CD is of considerable interest with regard to the immune system. Stat3 knockout mice are not compatible with life, but conditional knockout in myeloid cells (in those expressing lysozyme M) results in elevated Th1 activity and chronic enterocolitis (294). Similarly, Stat3 deletion during hematopoiesis leads to CD-like disease and lethality, with enhanced LPS-induced NF-κB-activation (295). Furthermore, Stat3 silencing in CD11c-expressing DCs is sufficient for the development of CD-like disease, with DCs showing defective suppression of LPS-induced activation (118). On the other hand, in humans the clinical picture of dominant-negative STAT3 mutations is that of hyper-IgE syndrome: a state of immunodeficiency that is interestingly characterized by poor Th17 differentiation (296).

However, in human subjects with CD, enhanced STAT3 phosphorylation or protein or mRNA expression has been reported in intestinal T cells (297-299) and in unfractionated biopsies (300,301). Discrepant results have been obtained regarding alterations of STAT3 phosphorylation in the peripheral blood T cells (297,299). Importantly, in a study by Musso and colleagues it was observed that enhanced

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STAT3 phosphorylation in intestinal T cells and macrophages was confined to the areas of active inflammation (299). In pediatric CD, elevated basal pSTAT3 levels in peripheral blood granulocytes and enhanced IL-6/sIL-6R-induced levels in CD4+ T cells have been described in comparison with healthy controls (302). Recently, the same group also found in pediatric CD patients that the CD-associated genetic variant of STAT3 (303) predisposes to increased levels of pSTAT3 after IL-6/sIL-6R-stimulation in blood-derived granulocytes (304). Experimental blockade of IL-6 trans-signaling (i.e., signaling by IL-6 + sIL-6R) has also been demonstrated to revert the resistance of mucosal T cells to apoptosis, alleviating intestinal inflammation in mouse models (305), and the antiapoptotic effect of IL-6/STAT3-signaling is appreciated as a disease mechanism in human CD (306). It is also likely that the enhanced STAT3-phosphorylation in the intestinal T cells in CD is related to the expansion of Th17 cells (306-308) and enhanced IL-6 production have been reported by both intestinal T cells and macrophages from CD patients (305). However, STAT3 is also needed for Foxp3+ Treg -mediated inhibition of colitis, associated with Th17 control (309). Taken together, it seems that the effects of STAT3-mediated signaling on intestinal inflammation, already within the cells of the immune system, are diverse and dependent on the cell type in question.

In addition to STAT3, one of its main activators, IL-10, is of key importance in the protection against gut inflammation. This is seen in Il10 knockout mice (310), in patients with mutated IL-10R that develop an aggressive IBD-like disease very early in life (18), and evidenced also by the association of IL10 to IBD in GWASs (18). Furthermore, human subjects with severe phenotypes of CD show deficient IL-10 production, also by DCs (311). 2.6.2.2 CD and experimental intestinal inflammation – viewpoint of T cells CD was considered a Th1-mediated disease, driven by an overproduction of IL-12, largely because of the well-established role of the p40 subunit of IL-12 in both various animal models and human disease, although the direct inhibition of IFN-γ was effective only in very early phase of the disease in animal models (312,313). However, GWASs implicated IL-23R in human CD, and it was also experimentally demonstrated that the p40-dependency of intestinal inflammation is actually related to the requirement of another heterodimeric cytokine, IL-23, which shares the p40 subunit with IL-12; the IL-23-specific p19 subunit - not the IL-12(p70)-specific p35 - was needed for the disease phenotype both in systems with intact (314) and deficient (315) adaptive immunity. This was consistent with observations from anti-IL-12p40-treated patients; IL-23 was also downmodulated, together with IL-12p70 (316). However, rather contradictory results were obtained from TNBS-induced colitis model, where IL-23-deficient mice were highly susceptible (317) (this is of interest because this model does not preclude any leukocyte subset, e.g., the CD8+ CTLs). The involvement of IL-23 in Th17-immunity, combined with elevated IL-17 levels,

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increased percentages of IL-17+ T cells, and upregulated IL-23 in IBD patients (308,318-320), together with the experimental demonstrations of the contribution of IL-17 in intestinal inflammation (321), led to the notion of CD as a disease mediated by both Th1 and Th17 cells (16). On the other hand, the Th17-related cytokine IL-22 has mostly been attributed with protective properties, although it could be pathogenic in some circumstances (16).

It has been shown in mouse models that the Th17 phenotype, and also its balance with that of Treg, is connected to, and may be controlled by, certain commensal bacteria and the balance between the species (292,293,322). However, numerical defects of Foxp3+ Tregs have not been readily demonstrated in human CD. On the contrary, elevated numbers and intact suppressive potential have been reported in intestinal tissues of patients (323,324). Nonetheless, it is intriguing that Foxp3+ Tregs from the intestinal mucosa of CD patients, but not from control subjects, exhibited IL-17 production (325), which may be indicative of plasticity-associated functional alterations between the interrelated Treg and Th17 phenotypes. Interestingly, enhanced IL-10 production by LP CD4+CD25+ cells was demonstrated in patients with CD-related condition ankylosing spondylitis in comparison with both CD and healthy controls, raising the possibility that the ability to enhance the IL-10-mediated regulation by Tregs keeps the intestinal inflammation in check in these individuals, preventing CD (324).

Indeed, T cell-related IL-10 signaling appears to be of central importance in the intestinal immunoregulation. It has been experimentally demonstrated that Tr1 cells suppress Th17 cells and IFN-γ/IL-17 double producers (Th17/Th1), and that the suppression of Th17 and Th17/Th1 by Foxp3+ Tregs is mediated by IL-10, and that the inhibition of colitis by these Treg subtypes is IL-10-dependent (326). Notably, Foxp3+ Tregs themselves need IL-10-mediated STAT3 activation in order to suppress Th17-associated intestinal inflammation (327). The concept of IL-10 and TGF-β-mediated control of Th1 responses has also been demonstrated in human colon samples (328). Furthermore, based on data from the CD4+CD45RBhigh-transfer model of colitis with RORγtgfp/+ knock-in mice, Tregs influence the balance of Th17, Th17/Th1 and Th1-like phenotypes (differentiated from Th17-related transcription factor RORγt-expressing cells) inhibiting Th1 development and transition, which coincides with the suppression of colitis (159). Accordingly, and perhaps similarly to T1D, by developing independently of classical Th1 cells (159), the Th17 – possibly originally Treg (156) – phenotype may sometimes, in a sense, act like a Trojan horse, sneaking in TCR specificities that become pathogenic when matched with the Th1-like effector arsenal. One suggested mechanism how the transition from Th17 to Th1, and associated acquisition of pathogenicity, takes place in the context of gut inflammation is via IL-17-induced IL-12 and IL-23 production from DCs (329).

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2.6.2.3 DCs in CD and in experimental intestinal inflammation If the balance between the regulatory and effector mechanisms of the immune system is critical to prevent CD-like inflammation, what is then known about the direct contribution of DCs? As discussed above, in mouse models intestinal CD103+ DCs and CX3CR1+ DCs/macrophages have been characterized with distinctive properties, the former inducing readily Foxp3+ Tregs (189). In a seminal paper by Annacker and colleagues in 2005, CD4+CD25+ Tregs failed to prevent CD4+CD45RBhigh-transfer colitis in CD103-deficient recipients, and non-T cell CD103 expression in competent animals occurred mostly in DCs (213). It was however later demonstrated that the development of intestinal inflammation reverts the ability of the MLN-derived CD103+ DCs to induce Foxp3+ Tregs in vitro (330). Hence, it could be that no dominant protection can be conferred by this DC subtype (330). The production of IL-10 by APCs has been mechanistically shown to be involved in the control of Th1 and Th17 responses to commensals and in the inhibition of colitis induced by an adoptive transfer of CD4+ T cells (331). When reconstituted exclusively with monocytes, mice were highly susceptible to DSS-induced (innate) colitis mediated by TNF secretion by CX3CR1+ DCs (29). Of note, high-level IL-23 production in mouse intestine is attributed to CD11c+ DCs both in inflammation and homeostasis (315,332).

In human CD, DCs and macrophages were in the late 1980s described in the inflamed human intestine in both CD and UC, with immunophenotypic alterations in comparison with healthy individuals (333,334). As discussed in previous sections, the distinction between macrophages and DCs is not clear at present. This is especially so in the gut, and it seems that some of the earlier macrophage-related observations could have involved cell types now more commonly considered DCs. Notably, macrophages isolated from human intestinal LP expressed DC-related markers, exhibited potent ability to activate allogeneic T cells in vitro, and in CD patients induced more IL-17+ cells than in controls, both unstimulated and with bacterial stimulus (335).

Increased total and mature mDC numbers (DC-sign+/CD83+/fascin+ cells) have been observed in human colonic samples from CD patients using immunohistochemistry, with an elevated expression of CD80, IL-12 and IL-18 in the DC-sign (CD209)+ cells (49,221,336). However, also a reduction in the colonic DCs, identified with anti-CD11c +/- anti-CD83 Abs, was reported and discussed to be in part due to corticosteroid treatment (337). Nevertheless, in another study the vast majority of patients was also on steroids, and still exhibited increased colonic DC numbers (221). However, it is possible that the use of anti-DC-sign or anti-CD83 Abs to identify DCs represent somewhat unspecific methods, as discussed (49,52). In the peripheral blood of CD patients, decreased numbers of mDCs and pDCs have been found, especially so in active disease (338). Enhanced phenotypic activation of mDCs has been reported in both active and inactive disease in cultured blood-derived mDCs, both with and without LPS-stimulation, and also in the inflamed colonic tissue, in the

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form of elevated CD40 expression (52,339). In a study of Baumgart and colleagues, CD1c+ mDCs were also numerically increased in the inflamed colonic mucosa in comparison with control subjects. In blood-derived mDCs, LPS-induced TNF production in remission and IL-8 production in active disease were also significantly elevated, as was TLR4 expression at the mRNA level in active disease (52). de Baey and co-authors found an increased frequency of M-DC8+ monocytes/DCs, potent producers of TNF but poor producers of IL-10, in both ileum and blood of CD patients, and reported that these cells disappeared from the ileum upon steroid treatment (340). In MLNs of CD patients, elevated numbers of CCL19 (a ligand for CCR7) positive mature DCs have been demonstrated in comparison with UC patients (341).

Directly relating human intestinal DCs to enhanced Th1/Th17 responses, Sakuraba and colleagues demonstrated in active CD enhanced IFN-γ and IL-17 responses in MLN-derived CD4+ T cells, accompanied by an elevated IL-23 production from Enterococcus faecalis extract-stimulated mDCs and “mature” DCs isolated from the MLN (in comparison with UC). Of note, IL-10 production was lower in CD. In allogeneic MLR, mDCs from the MLNs of CD patients induced enhanced IFN-γ responses, which were further augmented by the addition of exogenous IL-23. mDC/pDC-ratio in the MLNs was also increased (320). DCs (CD1c+) in the LP have also in humans been demonstrated to produce IL-23, although rather unexpectedly upon activation with a TLR8(/7) ligand resiquimod (especially in the additional presence of LPS) (53).

It is now relatively well established that human IECs promote tolerance in DCs in vitro, involving the induction of CD103-expressing DCs. Originally, the effect of epithelial cells in the human system was demonstrated as a strong DC-induced Th2 bias even in the presence of a potent Th1 induction (342). With human moDCs, this mechanism have been shown to be at least in part dependent on TGF-β, retinoic acid, and TSLP (30). Importantly, IECs from CD patients showed reduced transcription of TSLP and TGF-β1, combined with an impaired induction of Foxp3 in allogeneic naive T cells cocultured with the IEC supernatant-exposed DCs (30).

Type I IFN is protective against intestinal inflammation in several mouse models. This has been shown especially in relation to TLR9-induced signaling elicited by bacterial DNA (343), but also with imiquimod stimulation of TLR7 (344). It was mechanistically proven in a T-cell transfer model that the protective effect of CpG is mediated by CD11c+ DCs, and pDCs (in mouse CD11c+) are especially implicated (345). In human CD, Baumgart and associates recently carried out a pDC-focused study (220), in which pDC numbers were significantly increased in the colonic mucosa and MLNs of patients with an active disease - in some disagreement with earlier reports that failed to find any increase in CD (49,221). Furthermore, blood-derived pDCs showed elevated spontaneous production of TNF, IL-6, and IL-8, but, importantly, attenuated IFN-α response upon stimulation with CpG. Cultured blood-derived pDCs from patients with an active disease also expressed higher levels of CD40. Finally, it is intriguing that a clear increase of CD123+ putative pDCs was

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detected in colonic mucosa in most patients responding to treatment with G-CSF (a growth factor that favors pDC hematopoiesis over that of mDCs (37)), which coincided with a significantly enhanced IL-10 production by circulating CD4+ memory cells (39).

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3 Aims of the study The overall aim in this thesis was to explore possible alterations of APCs, particularly those of DCs, in T1D and CD, which are both diseases characterized by defects in the regulation of T-cell responses. The specific aims in the studies I-IV were to: I. investigate whether an abnormal activation of IL-17 immunity occurs in

pediatric T1D, and if so, whether it is accompanied by an altered activation of APCs

II. characterize the phenotypic and functional properties of circulating DCs at the

diagnosis of T1D III. characterize cytokine-induced STAT3 and STAT1 signaling and

immunophenotypic activation status of blood-derived DCs of CD patients IV. investigate in CD whether DCs directly contribute to the altered Th17

activation seen in patients

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4 Materials and methods 4.1 Study subjects In study I, pediatric patients with a relatively recent onset or newly-diagnosed T1D were investigated. Control subjects were nondiabetic healthy children. In the monocyte substudy, control children were age-, sex-, and HLA-risk genotype matched nondiabetic siblings of T1D patients without signs of β-cell autoimmunity. Study II comprised newly diagnosed pediatric patients with T1D, children at risk, i.e., siblings of T1D patients positive for ≥2 AAbs, and siblings without signs of β-cell autoimmunity. Siblings of diabetic patients did not belong to the same family with the diabetic individuals studied in a given series.

CD patients in studies III and IV were adults who were recruited on endoscopic-follow up. The blood sample was generally taken within three days of colonoscopy. The control groups comprised volunteers free of autoimmune disease or acute illness, and in study IV also patients who underwent endoscopy due to non-inflammatory indications without inflammatory findings. Disease phenotype and localization were defined in accordance with the Montreal classification. Endoscopic disease activity was evaluated using the Simple Endoscopic Score for Crohn’s disease (SES-CD), and Crohn’s Disease Activity Index (CDAI) was used as a measure for clinical activity.

See Table 1 and studies I-IV for more information. 4.2 Laboratory methods 4.2.1 Isolation of T cells and APCs Prior to all cell-isolation procedures and culture experiments, peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples or from blood-donor buffy coats by density-gradient centrifugation using Ficoll-Paque solution (GE Healthcare, Uppsala, Sweden). PBMCs were then either immediately cultured as such, fractionated to subpopulations, or alternatively frozen according to standard protocols and thawed prior to use.

Cell isolations were performed by magnetic sorting with MACS equipment and reagents (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions. For the comparison of Th17-related gene expression in CD4+ memory T cells between T1D patients and healthy children, CD45RA-expressing naive T cells were depleted from the purified CD4+ fractions using CD4 Multisort Kit and CD45RA microbeads. In CD studies, naive, memory, or total CD4+ T cells were negatively isolated using appropriate kits to label undesired cell populations with

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Table 1. Summary of study subjects. Study Analysis Patients Control subjects I cytokine and transcription

factor expression in anti-CD3/CD28-stimulated PBMCs

children with T1D: newly diagnosed or with a relatively recent onset, n=15

healthy nondiabetic children, n=12

cytokine and transcription factor mRNA expression in CD4+ memory T cells

children with T1D: newly diagnosed or with a relatively recent onset, n=9

healthy nondiabetic children, n=8

IL-1β and IL-6 mRNA expression in monocytes

children with newly diagnosed T1D, n=8

AAb-negative age-, sex-, and HLA risk genotype-matched siblings of unrelated T1D patients, n=8

II DC phenotyping and enumeration

children with newly-diagnosed T1D, n=16

age- and sex-matched siblings of T1D patients, negative for biochemically determined AAbs, n=15

mRNA expression analyses in mDCs

children with newly-diagnosed T1D, n=10

age- and sex-matched AAb negative siblings of T1D patients, n=10

flowcytometric assessment of cytokine-induced NF-κB, STAT1 and STAT3 phosphorylation

newly-diagnosed patients with T1D, n=13

AAb (≥2) positive age-, sex-, and HLA risk genotype-matched siblings of T1D patients, n=13

AAb negative age-, sex-, and HLA risk genotype-matched siblings of T1D patients, n=13

III flowcytometric assessment of cytokine-induced STAT1 and STAT3 phosphorylation, DC phenotyping and enumeration

adults with CD of variable duration, n=22 (n=18 for STAT studies; n=18 for phenotyping and enumeration)

healthy volunteers free of autoimmune disease or acute infection, n=21 (n=14 for STAT studies; n=18 for phenotyping and enumeration)

cytokine transcription in pDCs

as above, n=11 as above, n=11

studies on the effects of IL-6 on pDCs

- as above; pDC maturation by flow cytometry (n=5), pDC mRNA expression (n=9; 2 shared samples with the “pDC maturation” series), pDC-T cell-MLR (n=8), IL-6R-related studies (n=6)

IV LPS- and PGN-induced moDC activation and the induction of memory cell Th17 responses by moDCs

as above, n=17 n=15; generally healthy laboratory personnel (n=9) and patients without IBD (or malignancies) who underwent endoscopy without inflammatory findings (n=6)

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biotin-conjugated mAbs, followed by their depletion with anti-biotin microbeads. Monocytes were isolated making use of the same principle, whereas CD1c+ mDCs were purified by positive selection after depletion of B cells (which express CD1c as well). pDCs were isolated by the depletion of non-pDCs, which in study III was combined with a positive selection with anti-BDCA-4 microbeads when obtaining highly purified cells for mRNA transcription analyses. For more details, e.g., the purities achieved in various protocols, see studies I-IV. 4.2.2 Cell-culture experiments RPMI 1640 culture medium (Gibco/Invitrogen, Carlsbad, CA, USA/ Life Technologies, Paisley, UK), supplemented with 25 mM HEPES and 2 mM of L-glutamin, was used in all series for blood-derived cells, with some modifications regarding the added serum: 5-10% of heat-inactivated human AB serum was used with PBMC and mDC cultures and in moDC stimulation; 10% heat-inactivated FCS was present in pDC- and pDC + T cell-cocultures, and during moDC differentiation. Either gentamicin (study I) or penicillin + streptomycin (studies II-IV) were used as antibiotics. In some experiments, 50 µM 2-mercaptoethanol (2-ME) was also present to improve viability. Round- or flat-bottom 96-well plates (Nunc, Roskilde, Denmark) were used in the cell cultures, except in moDC-differentiation (48-well plates; Nunc) and in flowcytometric phosphorylation studies (loosely capped 5 ml tubes; BD Biosciences, San Jose, CA, USA).

PBMC cultures were performed in study I to compare the levels of Th17-related markers after T-cell stimulation with plate-bound anti-CD3 mAb (wells were coated with 5 µg/ml of anti-CD3) and 0.5 µg/ml of soluble anti-CD28 mAb (clones UCHT1 and CD28.2, respectively; both from BD Biosciences) for 40 h. Short-term (2.5 h) cultures of frozen and thawed PBMCs were also used in study II for the induction of NF-κB, STAT1, and STAT3 phosphorylation by a 10-minute incubation with 20 ng/ml IL-1β, 1000 IU/ml IFN-γ, or 100 ng/ml IL-10 (all from Peprotech, Rocky Hill, NJ, USA), respectively. Cells were then immediately fixed for 10 minutes by adding paraformaldehyde (PFA) in PBS to a final concentration of 1.75% (w/v) at room temperature (RT).

mDCs were cultured for 16-18 h to elucidate changes in the expression levels of CCR2 induced by TLR4-ligand LPS (E. coli 0111:B4; Difco, Detroit, MI, USA), TLR7/8-ligand CL097 (InvivoGen, San Diego, CA, USA), or IL-1β (R&D Systems, Minneapolis, MN, USA), or by the CCR2 ligands MCP-1 (CCL2) or MCP-3 (CCL7) (both from R&D Systems) and the effect CCR2 ligands was also assessed at 2 h. pDCs were cultured with IL-3, in the presence or absence of type B CpG (InvivoGen) and/or IL-6, for 16 h (RT-qPCR measurements) or for 2 d when used for flowcytometric assessments or as activators of allogeneic naive CD4+ T cells. moDCs were differentiated from monocytes in the presence of 50 ng/ml of GM-CSF

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(Peprotech) and IL-4 (R&D Systems) for 5 d, and then stimulated with 100 ng/ml of LPS or 10 µg/ml of PGN (E. coli 0111:B4; InvivoGen), or left untreated, for 18 h.

Purified naive CD4+ T cells were activated with the variably treated allogeneic pDCs for 6 d at a pDC-T cell ratio of 1:5 and then restimulated with plate-bound anti-CD3 and soluble anti-CD28 mAbs (anti-CD28 now at 1 µg/ml) for 16 h, when evaluating the effect of IL-6 on pDC function. Alternatively, CD4+ T cells were activated for 6 d with anti-CD3/CD28-coated beads in the presence of moDC-culture supernatans from CD patients and control subjects and then restimulated for 4 h (RT-qPCR) or 5 h (flow cytometry) with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) with 1 µg/ml of ionomycin.

From all cultures of isolated blood-derived cells, supernatants were collected for cytokine analyses or for functional assays, and cells were either harvested for flowcytometric measurements or for functional studies, or they were lysed for quantitative real-time PCR (RT-qPCR).

In study III, short-term (1 h) whole-blood cultures were performed for the assessment of cytokine-induced STAT1 and STAT3 phosphorylation. Briefly, after 30-minute preincubation, DCs were labeled with the fluorochrome-conjugated mAbs and the following cytokines were then added for 10 minutes: 1000 IU/ml of IFN-α-2a (Ropheron-A, Roche), 1000 IU/ml of IFN-γ, 250 ng/ml of IL-10, or 50 ng/ml of IL-6 (all from Peprotech, Rocky Hill, NJ, USA). Erythrocytes were then lysed with FACS Lysing Solution (BD Biosciences), followed by further fixation of leukocytes by adding PFA solution prior to permeabilization and intracellular staining. Human pancreatic islets in study I were isolated in the Central Laboratory of the Nordic Network for Clinical Islet Transplantation, Uppsala, Sweden, as previously described (346). Following Ficoll gradient purification, the enriched islet fractions were shipped to Helsinki. Islets were incubated in nonadherent 24-well plates in RPMI 1640 medium, supplemented with 10% FCS and antibiotics. MIN6 cells (a mouse insulinoma cell line) were cultured in 12-well tissue culture plates in DMEM medium (containing 4.5 g/l glucose; Lonza, Verviers, Belgium) supplemented with 250 µM 2-ME, 15% FCS, and antibiotics. Both human and mouse cells were cultured either with (recombinant human) IL-17A (100 ng/ml; eBioscience, San Diego, CA), with IL-1β (5 ng/ml; Peprotech) + IFN-γ (50 ng/ml; Peprotech), with all cytokines, or left untreated, for 24 h (MIN6 cells) or 72 h (human islets). 4.2.3 Flowcytometric analyses Cell-surface molecule staining was performed either in whole blood or in suspensions of cells in staining/washing buffer consisting of 5% FCS in PBS supplemented with 0.02% (w/v) sodium azide. However, in study I 0.5% bovine serum albumin (BSA) was used in the buffer instead of FCS. Cultured cells were washed with the buffer prior to staining. Fluorochrome-conjugated mAbs were added for 20 minutes at RT, and cells were washed twice (in some analyses of cell-fraction purities only once)

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with staining/washing buffer. In whole-blood samples, erythrocytes were then lysed with FACS Lysing Solution prior to washing. If not intracellularly stained, cells were then suspended in 1% PFA in PBS, or alternatively in staining/washing buffer if 7-amino-actinomycin D (7-AAD; eBioscience) viability dye was used, or in sheath fluid (study I). For DC enumeration, total leukocyte count was determined in a routine hematology laboratory to enable absolute DC quantitation. For intracellular staining of cytokines, monensin-containing GolgiStop (BD Biosciences) was present during the last 4 hours of culture prior to washing and surface-marker staining of cells. In study IV, staining with an amine-reactive green fluorescent dye was performed for dead cell exclusion instead of surface-marker staining. Cells were fixed and permeabilized using Fix/Perm and Perm/Wash solutions (BD Biosciences) and then stained for 30 minutes at +4°C, washed, and suspended in 1% PFA in PBS or in sheath fluid (study I) for analysis. For the staining of the serine- or tyrosine-phosphorylated forms of NF-kB p65 (pS529), STAT1 (pY701), and STAT3 (pY705), fixed cells were spun down and permeabilized with -20°C methanol at a final concentration of 80% (v/v) on ice. Cells were then spun down again, washed twice with staining/washing buffer containing 10% FCS, and stained with the mAbs for 30 minutes in RT in the same buffer. After two washes with staining/washing buffer (5% FCS), cells were suspended in 1% PFA in PBS for the analysis.

Data were acquired with FACSCalibur or FACSAria flow cytometer (BD Biosciences). FlowJo software (Tree Star, Inc., Ashland, OR, USA) and in study I also FACSDiva software (BD Biosciences) were used for data analysis. For details of mAbs, isotype-matched negative controls, gating strategies, and fluorescence quantitation, please see studies I-IV. 4.2.4 Cytokine and chemokine measurements from serum, plasma, and cell-culture supernatant samples In study I, IFN-γ levels in culture supernatants were determined using an in-house ELISA assay, and IL-17 was measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA). Plasma IL-6 levels in study I were assessed with a Cytometric Bead Array (CBA) kit (BD Biosciences). MCP-1 (CCL2), MCP-3 (CCL7) and/or Flt3 ligand were analyzed in serum (study II) or plasma (study III) samples using components from Milliplex MAG Human cytokine/chemokine kit (Millipore Corporation, Billerica, MA, USA) with Magpix system and xPonent software (Luminex Corporation, Austin, TX, USA) and with Prism 5 software (GraphPad Software, La Jolla, CA) using five-parameter logistic curve-fitting method. The remaining assessments were performed with FlowCytomix Multiplex Technology kits (Bender MedSystems, Vienna, Austria, and eBioscience) according to the manufacturer’s instructions. Data were analyzed using FlowCytomixPro

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software (eBioscience) and Prism 5 software. FACSCalibur flow cytometer was used for all cytometry-based immunoassays. 4.2.5 RT-qPCR mRNA expression analyses were performed with quantitative RT-PCR. Cells were lysed with the appropriate lysis buffer supplied with the RNA isolation kit, and the lysates were stored at -80°C. Total RNA was either isolated with RNeasy Micro plus kit, RNeasy Micro kit (monocytes in study I), or RNeasy Mini kit (PBMCs and human islets in study I) from Qiagen (Hilden, Germany), or with Mammalian Total RNA kit (memory cells in study I; Sigma-Aldrich, St. Louis, MO, USA). cDNA was synthesized using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was carried out using TaqMan fast universal master mix with predesigned FAM–labeled TaqMan Gene Expression Assay reagents (Applied Biosystems) in triplicate wells (see studies I-IV for specific assays used). Ribosomal 18S RNA was used as an endogenous control. The sequence detector (Stepone plus real-time PCR systems, Applied Biosystems) was programmed for an initial step of 20 seconds at 95°C, followed by 40 thermal cycles of 1 second at 95°C and 20 seconds at 60°C.

An exogenous cDNA pool calibrator was collected from phytohemagglutinin (PHA)-stimulated PBMCs and served as a standard to which normalized samples were compared (except for IL-4 measurements where a pool of anti-CD3/CD28-stimulated CD4+ memory cells was used as a calibrator, and in islet cell studies where RNA from human islets was used). Target gene expression was quantitated by the comparative threshold cycle (Ct) method: ΔCt is the difference between Ct of the target gene and Ct of the 18S gene, whereas ΔΔCt is the difference between the ΔCt of the sample and ΔCt of the calibrator; 2-ΔΔCt then gives the relative amount of the target gene in the sample compared with the calibrator, both normalized to the endogenous control. In T-cell analyses in study III, ΔΔCt was calculated as a difference between the ΔCt of a given sample and the highest ΔCt in that series of samples (the sample of lowest expression thereby getting a relative expression value of 1). 4.2.6 Determination of islet cell viability and apoptosis Nuclear staining with 5 mg/ml of Hoechst 33342 (HO) and 5 mg/ml of propidium iodide (PI) (both from Sigma-Aldrich) was used to assess the percentages of necrotic and apoptotic cells in the samples. HO passes all cell membranes and stains DNA blue, whereas PI can only enter necrotic and late apoptotic cells staining their DNA red; necrotic and apoptotic cells can then be detected by the white (HO + PI) nuclear staining and fragmented nuclei. Cells were stained with the dyes for 30 min at 37°C

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and then washed twice with PBS. Further dissociation with 0.05% trypsin and 0.02% EDTA was performed for the human islet cells, followed by cytocentrifugation to the slides prior to fixation, while MIN6 cells were directly fixed on coverslips with 4% PFA in PBS for 20 min at RT. Samples were examined with a fluorescence microscope and photographed for counting. 4.2.7 Diabetes-associated autoantibodies ICAs were assessed with indirect immunofluorescence on human group 0 donor pancreas. Specific radiobinding assays were used to measure IAA, GADA, IA-2A, and ZnT8A, as earlier described (240). 4.2.8 HLA genotyping PCR-based lanthanide-labeled hybridization method was used for the HLA typing of major T1D risk DR-DQ haplotypes, using time-resolved fluorometry for detection (347). 4.3 Statistical analyses Continuous data from independent samples in two groups were compared either with unpaired t-test or with Mann-Whitney U test (for non-normally distributed data), whereas paired samples were analyzed using paired samples t-test or Wilcoxon matched pairs test. Fisher exact test was used for the analysis when data were dichotomous (or dichotomized: CD4+ memory T cell mRNA transcription in study I; mDC NF-κB phophorylation in study III). Continuous data from independent samples in multiple groups were compared with Kruskal-Wallis test, followed by Dunn’s posttest. For dependent samples in multiple groups, repeated-measures ANOVA was used, followed by Dunnett’s posttest. For non-normally distributed data, Friedman test with Dunn’s posttest was utilized. Spearman rank correlation was used to examine the correlation between two parameters. A p value < 0.05 was considered statistically significant, except in the DC surface-marker screening in study II where the cut-off was at < 0.01 due to multiple testing. Two-tailed tests were used, except in the confirmatory RT-qPCR analyses in study II (one-tailed tests). Data were analyzed with Prism software (versions 5 and 6) or with PASW Statistics software (v18.0, SPSS Inc., Chicago, IL, USA).

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4.4 Ethical considerations The research was conducted in accordance with the Declaration of Helsinki. All participants or guardians of the children gave their informed consent, and assent was asked from children with ten years of age or older. Research was approved by the appropriate hospital ethics committees.

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5 Results and discussion – Type 1 diabetes 5.1 Children with T1D show signs of enhanced Th17 immunity In study I, we asked whether an abnormal activation of IL-17 immunity occurs in T1D as IL-17-producing cells are to some extent implicated in animal models of T1D (157,158,246,247) and clearly associated to certain immune-mediated diseases in humans (155). We found enhanced IL-17 secretion and IL-17A mRNA expression in T1D patients, in comparison with healthy children, in polyclonally (anti-CD3/CD28) T-cell-stimulated PBMC fractions, and IL-17A transcript expression was also elevated in purified unstimulated CD4+ memory T cells in an independent series of diabetic and healthy children (Fig. 3). Furthermore, increased transcription of IL-22 was detected both in anti-CD3/CD28-stimulated PBMCs (3.60 vs. 0.850 RU; p=0.005) and in the memory cells (5/8 T1D patients exceeding the highest observed level among the eight controls; p=0.026). mRNA expression of the Th17 transcription factor RORC2 was also increased in the stimulated PBMCs (70.1 vs. 19.3 RU, p=0.021), although RORC2 transcription could not be readily measured from the isolated memory T cells in most children (data not shown). mRNA expression for all markers was low or undetectable in the unstimulated PBMCs, with no differences between the groups. RORC2 and IL-17A transcript levels in anti-CD3/CD28-stimulation correlated with IL-17 secretion in T1D (ρ=0.599, p=0.018; and ρ=0.717, p=0.003, respectively). IFN-γ or T-bet mRNA expression did not differ between the groups of children studied (see study I), but IFN-γ transcription and protein production in anti-CD3/CD28-stimulated PBMCs nonetheless correlated with the IL-17 secretion in the diabetic children (ρ=0.652, p=0.006; and ρ=0.735, p=0.002, respectively). IL-17A + IFN-γ double producers within the CD4+ fraction were found with flow cytometry in four out of 11 diabetic children studied (we did not systematically study these double producers in the healthy children), their percentage being in the range of 15-35% among the CD4+IL-17A+ population (Fig. 4A). Although IL-17A + IFN-γ double producers are clearly induced also in healthy subjects (138), combined with our demonstration of the enhanced IL-17 immunity and the correlation between IL-17A and IFN-γ after polyclonal T-cell activation in T1D, this phenomenon may have pathophysiological implications. IFN-γ-producing CD4+ T cells generated from the RORγt+/IL-17+ background have been identified as highly pathogenic in animal models of inflammation and autoimmunity, also in the context of diabetes (157-159).

The immunophenotype of the IL-17A-producing cells in T1D patients was characterized using flow cytometry. First, IL-17A-producing cells in our culture conditions (the anti-CD3/CD28-stimulated PBMCs) were almost exclusively CD4+ (Fig. 4B). Second, all IL-17A-producing CD4+ cells were TCRαβ+ (Fig. 4C), and an overwhelming majority were positive for the CCR6 (median 95.2%, range 81.0-99.4%, N=7 children with T1D, Fig 4D). Hence, the enhanced IL-17A production we

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observed in T1D seems to be attributable to cells compatible with the reported phenotypic characteristics of Th17 cells (152). It is of interest that in the context of gut-associated mucosal immunity, CCR6 (expressed by the Th17 cells and Foxp3+ Tregs but not by Th1 cells) is involved in the fine-targeting and proper migration to certain anatomical compartments expressing the CCR6 ligand CCL20, and the deletion of CCR6 from adoptively transferred Th17 cells actually leads to an exaggerated intestinal inflammation, instead of being protective (348).

Of note, we detected also higher Foxp3 mRNA expression in T1D, when compared with the healthy children, both in anti-CD3/CD28-stimulated PBMCs (p=0.034) and in isolated memory cells (5/9 vs. 0/7; p=0.034). Interestingly, the Foxp3 levels in the diabetic children in the former series correlated with RORC2 expression and also with IL-17A secretion (ρ=0.846, p<0.001; and ρ=0.706, p=0.003, respectively). Furthermore, in the purified unstimulated memory cells, Foxp3 and IL-17A transcription were correlated (ρ=0.890, p<0.001). Indeed, Foxp3+ Tregs and Th17 cells are developmentally closely related (151), and it seems possible that the increased Foxp3 expression in T1D, and its correlation with the Th17 markers, may be related to an abnormally high level of phenotypic plasticity in patients. As discussed in chapter 2, the pathogenic population of IL-17- (and IFN-γ)-producing T-cells in the pancreatic islets of NOD mice harbors “ex-Treg cells” that have lost their Foxp3 expression and exhibit high pathogenic potential by rapidly causing disease upon adoptive transfer to NOD x Tcra-/- mice (156). Accordingly, if an elevated number of Foxp3+CD4+ T cells would exist in T1D, some of that population may start to behave detrimentally. Supporting this interpretation, Marwaha and co-authors found an increased frequency of Foxp3+ Tregs in T1D patients; intriguingly, this population included CD45RA-Foxp3low cells that exhibited no suppressive activity (349). Marwaha and colleagues also confirmed our finding of augmented IL-17 responses within the CD4+ T cell fraction by detecting IL-17+ cells by flow cytometry in pediatric T1D. They also reported an increased frequency of CD8+IL-17+ cells. A report by Arif and colleagues followed (350), in which T1D patients with a relatively recent disease onset were shown to have a dramatically increased frequency of CD4+

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cells that produce IL-17 in response to β-cell auto-Ags, thereby connecting the enhanced IL-17 immunity to β-cell autoimmunity. Th17 cells and Tregs have also been studied in the pancreatic-draining LNs from T1D patients (351), in which an increased percentage of IL-17+ cells within the CD4+ fraction was observed. In addition, the frequency of CD25brightFoxp3+ Tregs was decreased, accompanied by an attenuated potential to suppress pancreatic-draining LN CD3+ T cells. However, some caution is warranted as the control subjects in this study appear to be organ donors, and in suppression assays the responder CD3+CD25- T-cell numbers were not equalized and variable sources of monocytes were used to generate DCs for Ag presentation. 5.2 IL-17 augments the inflammatory and apoptotic effects of proinflammatory cytokines in pancreatic islet cells in vitro Having demonstrated enhanced Th17 activation in two independent series of children with recent onset T1D, we set out to elucidate the direct consequences of this alteration for the pancreatic β cells. To this end, human islets (N=6) were subjected to

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treatments with human IL-17A alone or in combination with the proinflammatory cytokines IL-1β and IFN-γ, and mRNA levels for some key β-cell stress or antiapoptotic factors were measured. IL-17A enhanced the transcription of (mitochondrial) superoxide dismutase (SOD2; p=0.04), and when combined with the proinflammatory cytokines, further increased the transcription of the inducible isoform of NO synthase (NOS2A, p=0.005) and COX-2 (p=0.02). The latter two mediators have been demonstrated to contribute to β-cell dysfunction and/or damage (352,353), whereas SOD induction may, however, counteract oxidative stress, exerting a protective effect by eliminating superoxide radicals (354). IL-17A treatment also down-modulated the mRNA expression for the antiapoptotic factor BCL-2 in human islets (p=0.04) and potentiated the proapoptotic effect of IL-1β and IFN-γ as observed directly by fluorescence microscopy in human islets (N=1). In MIN6 cells (N=3), this effect was also seen in the absence of IL-1β and IFN-γ (IL-17A vs. neg: p=0.03; IL-1β+IFN-γ+IL-17A vs. IL-1β+IFN-γ alone: p=0.04). The expression of IL-17RA and IL-17RC in human islets was confirmed by RT-qPCR (see study I for more details). Our observation of the apoptosis-promoting effect by IL-17 in combination with proinflammatory cytokines in pancreatic islets and murine β cells was confirmed in the study of Arif and colleagues, who also presented a case report of the expression of IL-17A, RORC, and IL-22 mRNA in cadaver islets obtained shortly after the diagnosis of T1D (350). The role of IL-22, which we found to be upregulated in T cells from recent-onset T1D patients, appears not to be sufficiently characterized in regard to islet cell function/homeostasis. However, the expression of IL-22 receptor in pancreas occurs especially in the islets, including the β cells (355). Given that IL-22 is known to exert a protective and proliferation-promoting effect in various non-immune cells within the gut and accessory organs (16), one could predict rather protective than debilitating impact in β cells. Moreover, especially because of this viewpoint, it would be valuable to further dissect the cell type to which the elevated IL-22 production is attributable (i.e., Th17 or Th22). In conclusion, the plausibility of the concept of direct β-cell toxicity by IL-17A secreted by the upregulated Th17 cells in T1D appears demonstrated. 5.3 Unaltered IL-1β and IL-6 transcription in APCs from diabetic children IL-1β and IL-6 are involved in the development of Th17 cells (135,147), and the latter also favors Th17 differentiation over the related (Foxp3+) Treg pathway (151). Therefore, in study I we measured the levels of IL-1β and IL-6 mRNA transcripts by RT-qPCR in unstimulated monocytes isolated from age-, sex-, and HLA-risk genotype-matched children (independent from the children studied for T-cell function). However Fig. 5A demonstrates that the levels of both cytokine mRNAs were similar between the groups. We also assessed the levels of IL-6 in plasma but found no difference between the diabetic and healthy children (from the children in

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the PBMC series in study I: 2.25 vs. 2.23 pg/ml, n.s.). In addition, we evaluated the spontaneous IL-1β transcription in purified mDCs but could not find any alterations in recent-onset T1D (Nieminen JK et al., unpublished results, Fig. 5C). Hence, contrary to the results of Bradshaw and colleagues (245), we could not demonstrate enhanced IL-1β and/or IL-6 production in patients with recent-onset T1D. One feasible explanation for this discrepancy could be the age of the individuals studied. In the report by Bradshaw and colleagues adults were also included, whereas our material was restricted to children with 5-9 years of age. As our controls in this substudy were siblings of unrelated patients sharing some T1D genetic predisposition, this may have diluted our findings. On the other hand, such control matching may help to dissect the most crucial disease-contributing factors from factors arising from shared background. Furthermore, enhanced spontaneous IL-1β and IL-6 transcription in monocytes has earlier been specifically connected to adult-onset T1D, in particular to LADA, rather than juvenile T1D (356). IL-1β transcription in childhood-onset patients with a long-standing disease, and interestingly also in their non-diabetic monozygotic twins, was even lower than in control subjects (357). Accordingly, it seems that pediatric T1D may represent an entity somewhat distinct from the adult-onset forms of autoimmune diabetes, not frequently associated with a proinflammatory cytokine-related gene expression profile in monocytes, but perhaps characterized, as suggested, by an altered expression of genes related to other functions, such as chemotaxis (356). In conclusion, Th17 expansion in pediatric T1D at the onset is not readily explained by an altered spontaneous production of IL-1β and/or IL-6 in APCs. Of course, this does not rule out the possibility of their altered activation upon stimulation.

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5.4 Numbers of circulating CD1c+ mDCs and pDCs are decreased in recent-onset pediatric T1D Altered numbers of circulating DCs have been reported in T1D, but results have not been consistent. Therefore, in study II we performed blood DC enumeration in a strictly defined cohort consisting of pediatric patients with recent-onset disease (n=16) and age- and sex-matched control children (n=15; these children were siblings of unrelated T1D patients). The blood sample was taken from the patients within 4-7 d from the diagnosis, except in two cases (1 d and 60 d from the diagnosis). Control siblings were free of biochemically-defined AAbs (two tested positive for ICA). We observed a decrease in both absolute (cells/µl blood) and relative (% leukocytes) mDC counts, and the absolute pDC counts were also significantly lower in patients (Fig. 6A and 6B). These findings did not correlate with the levels of plasma glucose or blood pH at diagnosis (data not shown), suggesting that the phenomenon was probably not due to hyperglycemia or ketoasidosis. In patients with the DQB1*0302/x genotype (n=9/16; “x” stands for a neutral haplotype) the absolute mDC and pDC counts were significantly lower than in other patients (p=0.031 and p=0.021, respectively), while no difference was seen in the control children (n=6/15). We did not find any difference in the levels of DC-growth factor Flt3 ligand between the patients and controls, the concentrations being, however, below the detection limit in most children (data not shown).

Our finding of reduced counts of both mDCs and pDCs is in accordance with an earlier study performed with a considerable number of children with a newly diagnosed T1D and appropriately age-matched controls (270). In other studies, increased (268), unaltered (269,273), and either decreased (271) or proportionally increased (272) pDC counts have been reported. However, in all of these reports

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either a mixed population of children and adults were studied, or the age of the study subjects was not disclosed. In one study, age was nonetheless controlled as a confounding factor (271). This could be a more valid approach as pDC counts have been reported to decline with age (84). Furthermore, the variation of disease duration in these studies has been considerable, and the duration of disease defined as recent-onset has also been up to 6 months. Potential reasons for the discrepant findings may include methodological differences as well, such as strategies in flowcytometric DC identification and the choice of reagents in it. These seem to influence fluorescence intensity levels, likely affecting the sensitivity and specificity of the assays used.

Alkanani and co-authors more recently compared AAb+ individuals (both children an adults) free of diabetes with AAb- controls (358). However, no differences in the relative counts of CD14+ monocytes, CD1c+ mDCs, or pDCs were observed. Hence, despite the aforementioned potential caveats in the interpretation, it could be that the prediabetes phase is generally not characterized by numerical alterations of APCs. Nevertheless, it is intriguing that reduced yield and maturation of DCs from monocyte precursors has also been reported in T1D and in individuals at risk (274,275,277,278), a finding that could be seen as in concordance with ours. 5.5 Expression of CC-chemokine receptor 2 (CCR2) and activation of NF-κB pathway in peripheral blood DCs in T1D The phenotypic properties of DCs have only been superficially characterized in T1D, the literature being limited to the most obvious maturation markers. To address this issue, we carried out a broader flowcytometric screening of DCs, comprising 20 markers deemed potentially relevant surrogates for the functional aspects of microbe recognition, chemotaxis, adhesion, Ag presentation, T-cell costimulation, and immunoregulation. We assessed both the median fluorescence intensities (MFIs, with the negative-control mAb intensity subtracted) and the percentage of cells expressing given molecule above the negative-control mAb level. The significance cut-off for the screen was set at p<0.01 to take into account the multiple-test setting. Alterations achieving statistical significance were: 1. reduced expression of the chemokine receptor CCR2 in both mDCs and pDCs in T1D (Fig. 7A and 7C); 2. decreased expression of the immunoregulatory CD200R molecule (median MFI 1.48 vs. 2.49 AU, p=0.005; 15.3 vs. 25.2%, p=0.004) on pDCs in T1D. Approaching statistical significance, also higher expression levels of DC-sign were observed in T1D (8.45 vs. 5.86%, p=0.013). See supplement for study II for a complete illustration of expression levels for all markers.

Following the initial flowcytometric screen, samples from an independent series of patients and control children were used for verification by RT-qPCR. The reduction in CCR2 expression could be confirmed in isolated mDCs (N=10 patients + 10 control subjects, Fig. 7B), whereas no difference was found in DC-sign mRNA levels (N=7+7; data not shown). In enriched pDC fractions from diabetic or control

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children, CD200R mRNA transcripts were not readily detectable by RT-qPCR (N=10 patients and 10 control subjects; data not shown). Furthermore, the intensity of CD200R staining on pDCs analyzed by flow cytometry was low even in healthy children, rendering this finding somewhat unreliable. CCR2 expression levels, measured either by flow cytometry or RT-qPCR, did not correlate with plasma glucose or blood pH at diagnosis, and the altered expression was not associated with particular HLA-risk genotypes either (data not shown). To seek further for possible explanations for the decreased CCR2 levels, we measured the serum concentrations of the main CCR2 ligands, given that elevated levels of MCP-1 (CCL2) have been described in long-standing T1D (359) and inhibition of CCR2 expression by exogenous MCP-1 has been reported in human myeloid cells (360). However, no differences were observed in the levels of MCP-1 or MCP-3 (CCL7) between the groups (median 451 vs. 380 pg/mL, n.s., and 8.35 vs. 16.6 pg/mL, n.s., respectively). We also measured the levels of MCP-1 mRNA transcripts in isolated mDCs by RT-qPCR, in order to exclude the possibility of an enhanced

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autocrine production, but could not demonstrate any difference (mean expression 152 vs. 148 RU, n.s.). Having confirmed that CCR2 expression on mDCs is profoundly downregulated by agents inducing mDC activation/maturation (see study II), we asked whether an altered NF-κB activation could be detected in APCs from patients and from children in risk. When activated with IL-1β, children with T1D and/or β-cell autoimmunity indeed showed a trend for an enhanced NF-kB p65 phosphorylation response in mDCs (p=0.063; Fig. 8). mDCs from patients and AAb-positive children exhibited similar distribution, with some individuals showing high-level reactivity (Fig. 8, p=0.160 when compared alone with the AAb-negative children). We also

measured phosphorylated forms of STAT1 and STAT3 after a stimulation with IFN-γ or IL-10, respectively, but could not demonstrate any differences between the groups of children studied. No significant differences were seen in the spontaneous phosphoprotein-staining intensities (with the negative-control mAb intensity subtracted). Earlier studies have demonstrated alterations of NF-κB signaling in T1D. Mollah and colleagues (279) found decreased LPS-induced late-phase nuclear binding of NF-κB proteins, which was accompanied by a reduced CD40 and MHC class I upregulation, but with normal production of IL-1β, TNF, IL-12p70, and IL-10. However, activation of p65 (RelA) and RelB was constitutive in monocytes from patients, and NF-κB inhibitor SHP-1 was overexpressed. Importantly, experimental inhibition of SHP-1 reversed the poor LPS-induced NF-κB binding in the differentiating DCs and improved their CD40 upregulation in most patients, while controls did not respond. Accordingly, the earlier findings of impaired DC

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differentiation may in part be due to an aberrant function of the NF-κB pathway. In human peripheral-blood primary CD1c+ mDCs, enhanced LPS-induced NF-κB p65 phosphorylation was observed in children and young adults with T1D of relatively recent onset (273). The trend for an increased IL-1β-induced NF-κB p65 phosphorylation, observed in our study in children with β-cell autoimmunity, suggests that the aberrancies in NF-κB in T1D may not be restricted to signaling from certain TLRs, but may instead affect a broader range of NF-κB-mediated signaling events. The report by Meyers et al. (273) also addressed the LPS-induced cytokine production of mDCs and monocytes, with slightly confusing findings: whereas patients had attenuated IL-6 responses in mDCs, IL-1β secretion from monocytes was increased (as was the IFN-α response in resiquimod-stimulated pDCs). However, given the diverse NF-κB-related alterations reported by Mollah et al. (279), this outcome may not be inconceivable. Indeed, when the same group more recently extended the study of TLR-ligand-induced cytokine responses to the prediabetes phase (358), it was found that individuals positive for AAbs (mostly for only one of two) exhibited an increased IL-1β response in monocytes for a wide range of TLR-stimuli, and also in mDCs for resiquimod. Also in prediabetes, IL-6 responses were instead attenuated, although here for polyI:C and resiquimod. Accordingly, it appears that whereas enhanced IL-1β responses seem to be associated with β-cell autoimmunity, with and without overt diabetes, IL-6 responses are rather attenuated in these individuals. If this is actually the case, it could be (assuming a disease-contributing role) that the phenomenon of differential IL-1β vs. IL-6 regulation may not necessarily be connected to APC-induced T-cell activation, but might also be related to direct effects on β cells by the cells of monocyte origin (8,241). IL-1β induces potentially detrimental NF-κB responses in the islets - including the upregulation of IL-17 receptor (350,361) – while IL-6 have been demonstrated to mediate not only destructive, but also protective effects on the β cells (362).

One can speculate that the findings of an enhanced NF-κB responsiveness and the decreased CCR2 levels observed in our study, are mechanistically interrelated, as CCR2 was downregulated by all NF-κB activators tested. However, no clear indication for any universal maturational alteration was observed (see study II and supplement). We could also not directly relate the reduced DC counts and CCR2 levels; the nature of the latter finding was not the absence of CCR2++ cells, but an overall reduction in expression. Furthermore, there was no correlation between these readings (data not shown). Thus, these results as a whole are not readily explained by an increased CCR2-mediated translocation of DCs to tissues, such as the inflamed pancreatic islets or pancreatic-draining LN. Nevertheless, it is intriguing that DC migration in response to MCP-1 is drastically impaired in NOD mice (363), which may indicate migration-related functional relevance for the decreased CCR2 expression in the context of autoimmune diabetes. This notion is even more interesting in the light of the relatively recent report (364), which demonstrated an unexpected partial protection in the NOD background when MCP-1 was overexpressed in the β cells under the rat-insulin promoter (RIP). This manipulation

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led to a 10-fold increase of CD11c+ DCs within the pancreatic tissue and in pancreatic LN, attributable to CD11c+CD11b+ subset that demonstrated a protective effect upon transfer to NOD recipient. Accordingly, it is tempting to speculate that the functional consequence of the reduced CCR2 expression on the DCs of human patients could be an impaired MCP-1-directed DC migration to the islets and pancreatic LN, and subsequently inefficient DC-induced tolerance. Although even more speculative, one could further conceive that if DCs cannot migrate normally, proportionally more self-Ag presentation would then take place by some other type of APCs, such as the B cells (which peak in the human insulitis in concert with the β-cell destruction (8) and are implicated as Ag-presenters for diabetogenic T cells (242)). However, some caution in the interpretation is warranted, as MCP-1 overexpression in the β cells actually proved harmful on a different (non-diabetes-prone) genetic background (365). Since CCR2 expression on DCs, or on DC precursors, is needed for normal Th1 responses (366), it seems plausible that our observation of CCR2 reduction could also be related to an impaired Th1 immunity, which could in turn predispose to the influence of certain infections (e.g., by enteroviruses).

Finally, it has been reported that intravenous infusion of insulin in patients with type 2 diabetes downregulates various markers associated with inflammation in PBMCs, including CCR2 (367), which opens the possibility that our CCR2 finding could have been due to exogenous insulin used by the patients. However, we did not observe in the patients any other changes associated with the intravenous insulin administration in this report, such as downregulation of CCR5 or serum MCP-1. Even if the rather dramatic reduction in the CCR2 levels on DCs would turn out to be a secondary phenomenon, it is still noteworthy that the diabetic individuals do bear this alteration, and it seems rational to think that it may have some immunological consequences that might be relevant (e.g., susceptibility to certain infections). In regard T1D immunopathogenesis, the logical next step would clearly be to study CCR2 in the prediabetes phase, also in cell types other than DCs.

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6 Results and discussion – Crohn’s disease 6.1 Decreased numbers of peripheral blood pDCs and BDCA3+ mDCs and changes in DC phenotype in CD Although DC-related research has also been carried out in human CD, many basic aspects, e.g., cell counts and the activation-related immunophenotype, have been previously assessed by relatively few studies, as discussed earlier. Fig. 9 illustrates our finding that both the absolute and relative counts of pDCs and BDCA3+ mDCs were decreased in CD patients (study III). CD1c+ mDC numbers were not significantly different from healthy controls, although a trend was seen for lower absolute (p=0.085) and relative (p=0.149) counts (Fig. 9). As in T1D, we measured the circulating levels of DC growth factor Flt3 ligand. Plasma concentrations above the detection limit (set at 16 pg/ml) were slightly more often encountered in CD, but not significantly so (11 out of 20 vs. 6 out of 18, p=0.210). Of note, the relative pDC and BDCA3+ mDC counts negatively correlated with the endoscopic disease activity measured by the SES-CD score (Fig. 9), while no significant correlation was seen for CD1c+ mDCs (ρ=-0.338, p=0.170). The observation of decreased pDC and BDCA3+ mDC counts, as well as their correlation with the disease activity, are in line with an earlier report by Baumgart and colleagues (338). Increased numbers of pDCs have recently been reported in the flaring colonic LP and MLNs from patients with CD (220), but this phenomenon has not been consistently observed (39,49,221). Thus, it is currently not clear whether the numeric reduction of blood pDCs is merely secondary to the intestinal inflammation. pDCs exert tolerogenic functions in numerous experimental settings by a variety of mechanisms (as discussed in more detail in chapter 2), including their propensity for Tr1-type of T-cell activation. We also found that pDC-primed CD4+ T cells from healthy donors produced high quantities of IL-10 upon restimulation, comparable to those of IFN-γ, which may signify importance for the reduced pDC numbers in CD. As discussed earlier, recent evidence suggests that T-cell IL-10 production is involved in the control of intestinal inflammation, also in relation to human CD (39,324,326). Another possible mechanism by which a pDC defect could contribute to the CD-related intestinal inflammation would evidently be via compromised supply of IFN-α – type I IFN is clearly protective in various experimental models of gut inflammation (343-345), and attenuated secretion of IFN-α has also been demonstrated in cultured pDCs from CD patients stimulated via TLR9 (220). However, in our study no spontaneous transcription of IFN-α could be demonstrated in highly purified pDCs either from patients or control subjects (N=11+11, data not shown).

The reduction in the numbers of the minor BDCA3+ mDC population has also been reported earlier (338). Fairly recently, more information has begun to accumulate regarding this subtype. It is now well established that this population is capable of highly efficient cross-priming, which has made it a prime candidate for a

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human equivalent of mouse CD8α+ mDC (70). Intriguingly, CD8α+ lymphoid tissue mDCs appear to be developmentally and functionally related also to peripheral tissue CD103+ DCs in mice (368), and the close resemblance between human BDCA3+ mDCs and mouse CD103+ DCs was also directly demonstrated in a very recent report (369). In an earlier study, BDCA3+ putative DCs were found in colon samples scattered throughout the LP, around the blood vessels in the submucosa and muscle layers, and also in the MLNs, with no gross numerical differences between the patients with CD and controls (49). Furthermore, highest expression of the gut-homing molecule CCR9 among human blood DCs has been attributed to this subtype (46). As discussed in chapter 2, BDCA3-expressing human mDCs may preferably induce both passive and active (i.e., Treg-mediated) tolerance in CD4+ T cells (71,72). These cells may also efficiently “cross-tolerize” CD8+ T cells, as their superior cross-presentation capacity is not necessarily contingent on PRR-mediated activation, as demonstrated (72). Although we could not subject this cell type to any functional testing and could only assess its phenotype in terms of HLA-DR expression (which

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did not show any alteration), the decreased count of the BDCA3+ mDCs in CD does thus represent an interesting finding. We also performed a rough activation/maturation-related immunophenotypic analysis of DCs (study III). It was found that pDCs exhibited decreased HLA-DR expression in CD patents, when compared with the healthy controls (median 430 vs. 531 AU, p=0.024), whereas CD40 expression on CD1c+ mDCs was higher in CD (median 0.625 vs. 0.435 AU, p=0.047). No differences were found in CD86 expression, and CD80 staining intensity remained at the level of negative-control mAb. Although fairly speculative, the decreased HLA-DR expression on pDCs may be functionally related to an altered pDC-mediated Ag-presentation and T-cell activation. In some disagreement with our results, Hostmann and co-authors (370) recently observed an increased expression of maturation markers in blood-derived pDCs from IBD patients when compared with healthy controls. However, the utility of this study is limited due to the pooled cohort of CD and UC patients used, especially given that the percentage of blood samples from CD patients was not disclosed. In addition, MLN pDCs were studied, but the IBD group included only a single CD patient. This study failed to find any difference in MLN pDC counts between the pooled IBD patients and controls.

The increased CD40 expression on mDCs, albeit slight in the present study, has been earlier described for in vitro cultured CD1c+ mDCs (52), and also for intestinal LP mDCs (52,339) in CD. It is readily conceivable that this alteration may predispose mDCs to initiate inappropriate T cell activation. This is particularly relevant considering the general Th1-preference by CD40-mediated costimulation (371,372) and that the CD1c+ subtype of mDCs appears superior in its T-cell activation capacity (45). In our study, it was found that the IL-10 signal - a potent inhibitor of DC activation (118) - was transduced more efficiently in the CD1c+ mDCs of patients (Fig. 10), which may thus represent a compensatory mechanism. 6.2 Altered cytokine-induced STAT1 and STAT3 phosphorylation in DCs from CD patients Although STAT1-, and in particular, STAT3-related findings have been reported in CD, to our knowledge, no prior studies have focused on DCs. As both STAT1- and STAT3-signaling are clearly implicated in DC regulation (see chapter 2.1.2.2), we performed in study III a flowcytometric single-cell-based evaluation of cytokine-induced phosphorylation of these signal transducers in blood-derived pDCs and CD1c+ mDCs. However, we did not observe any universal defect of STAT3 or STAT1 signaling. Our findings were instead cytokine-, and cell type-specific.

A pDC-restricted attenuation of IL-6-induced phosphorylation of STAT3 was seen in CD patients (Fig. 10). Notably, as reported (306), plasma IL-6 levels were higher in CD, detectable concentrations (>1.2 pg/ml) being observed significantly more often in patients (10/22 vs. 1/21, p=0.004), and the levels in patients also

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strongly correlated with the endoscopic disease activity (ρ=0.690, p<0.001), as demonstrated in earlier studies (306). However, the levels of IL-6-induced phosphorylated STAT3 (pSTAT3) in pDCs and mDCs did not correlate with the disease activity or with plasma IL-6 concentration, suggesting that the attenuated pSTAT3 response to IL-6 in pDCs is not explained by the elevated systemic IL-6. It

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may nonetheless be noteworthy that IL-6 downregulates MHC II expression in DCs (80), and HLA-DR levels were indeed decreased on pDCs of CD patients in our study. Interestingly, we did not see any alteration in the IL-10-induced STAT3 phosphorylation in pDCs, and the response was even enhanced in mDCs (Fig. 10). This not only implicates IL-6 signaling over that of IL-10 in pDCs in CD, but also suggests no reduction in the total cellular quantity of STAT3 protein that would explain the brighter staining with the phospho-specific mAb upon cytokine-induced phosphorylation.

IFN-α-induced pSTAT1 and pSTAT3 and IFN-γ-induced pSTAT1 were also assessed. In IFN-α-stimulation, both STAT1 and STAT3 responses were attenuated in both DC subtypes in CD patients, whereas the IFN-γ-induced pSTAT1 levels were not affected (Fig. 10). pSTAT1 and pSTAT3 staining intensities in the unstimulated samples (with the negative-control mAb staining intensity subtracted) were not significantly different between the groups (data not shown).

Type I IFN signaling plays a role in the regulation of DC activation; functional impairment of DC function is seen in its absence, including an attenuated activation of both CD4+ and CD8+ T cells, reduced uptake of Ag, and lower expression on MHC class II (94,373). However, it has been reported that some of these activities are dependent on the specific type of DC in question. For instance, IFN-α-induced upregulation of MHC class II was much greater in human mDCs, pDCs showing only a very mild effect (116). IFN-α is also a potent survival factor for both human pDCs and mDCs (116), and it is therefore possible that the attenuated IFN-α-induced signaling and the reduced DC counts are interrelated. In relation to our findings, it is also of interest that pDCs in mouse Peyer’s patches may be a functionally and developmentally distinct subset with a decreased capacity to produce IFN-α, but with a dependency on IFN-α/STAT1 signaling for their normal accumulation (374). Last, it is well established that IFN-α induces IL-10 secretion from human mDCs (116,375), which may link both the attenuated IFN-α-induced STAT-signaling and the possible numeric pDC defect to the impaired immunoregulation in CD. In mouse, the targeted silencing of Stat3 in DCs (in Stat3flox/flox × CD11cCre mice; note that mouse pDCs are CD11c+) leads to a CD-like ileocolitis (118). In these animals, DCs were not able to restrain the LPS-induced activation when given exogenous IL-10 in vitro. Another highly CD-implicated cytokine with STAT3-dependent signal transduction, IL-6, was not tested. One could expect a similar outcome (117), although there is some differential regulation, e.g., in the form of SOCS3 activity (119).

Since our findings from human CD pinpointed IL-6-induced STAT3 signaling in pDCs, we set out to elucidate its possible influence on the regulation of pDC activation, and on the T-cell activation preferences of pDCs. Although circulating IL-6 levels were generally low also in most patients, IL-6 is elevated in the inflamed intestine (305). It seems plausible that defective “feedback” to T-cell activating cells, such as the pDCs, may perpetuate inflammation. Unfortunately, we could not obtain sufficient numbers of cells from the patients for these functional experiments.

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Nevertheless, with cells isolated from healthy individuals, we observed that the phenotypic maturation of pDCs in culture with IL-3 was restrained in the presence of exogenous IL-6. However, stimulation with CpG reverted this effect, and CD40 expression was instead significantly increased by the IL-6 in the additional presence of type B CpG, a mimic for microbial DNA (see study III). More importantly, IL-6 treatment of pDCs, in the presence of CpG, enhanced IL-4 mRNA expression in 6 d cocultured and then restimulated naive allogeneic CD4+ T cells (p=0.003; see study III for illustration and more details). It also reduced the ratio of IFN-γ to IL-4, i.e., Th1/Th2 signature cytokine ratio, at the protein level irrespective of the presence of CpG during the pDC maturation (p=0.048 for CpG-; p=0.017 for CpG+), which was also seen at the mRNA level in the presence of CpG (p=0.006). Furthermore, IL-10 mRNA transcription after the coculture and T-cell restimulation was significantly increased by the IL-6 treatment of CpG-activated pDCs (p=0.034). IL-6 treatment of pDCs did not affect the viability of pDCs or T-cell proliferation (data not shown).

Given that CD appears as a disease characterized by an enhanced Th1 activity (16,320) and also by an insufficient T-cell IL-10 production (39,324,326), we speculate that the attenuated IL-6 signaling in CD patients’ pDCs may have functional consequences that are in line with these characteristics. Ogata and colleagues also recently confirmed (376) an earlier finding (88) regarding the suppressive effect of T cells primed with IL-3-activated pDCs (now with no additional stimuli), and further showed that the presence of anti-IL-6 mAb during the initial pDC-T-cell culture significantly attenuated the suppressive potential of the induced Tr1 cells. What is intriguing in relation to our findings, blockade of type I IFNs had similar influence when CpG-activated pDCs were used. However, the target cell (pDC vs. T cell) for these effects was not dissected in this setting, and IL-6 did increase ICOS expression on T cells (ICOS ligand-mediated costimulation by pDCs prefers the Tr1 phenotype (88)).

In a previous report, cultured blood-derived pDCs from CD patients showed elevated spontaneous production of IL-6, TNF, and IL-8 (220). This opens the possibility that the attenuated IL-6-induced STAT3 phosphorylation in our study could have been due to an enhanced autocrine cytokine production ex vivo. Therefore, we determined the levels of mRNA transcripts for IL-6 in highly purified (>99%) pDCs from patients (n=11, 73% with active disease, i.e., SES-CD score ≥4) and from control subjects (n=11). However, no amplification could be detected in the IL-6 RT-qPCR-assay; mRNA transcripts for the house-keeping gene 18S1 were detected at relatively high levels (at median of 13.7 RT-qPCR cycles), allowing the exclusion of IL-6 transcription in pDCs ex vivo with a theoretical upper bound of approximately 0.5 x 10^7-fold lower copy number in comparison with the house-keeping gene. Hence, we could not detect any signs for the production of IL-6 (or of IFN-α) in pDCs from either CD patients or control subjects (data not shown), which argues against an abnormally enhanced autocrine cytokine production ex vivo that would explain the difference in IL-6/STAT3-signaling in pDCs. Of note, DC-specific knockout of Stat3 also impairs STAT3-mediated suppression of IL-6 and TNF secretion (118). The

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attenuated IL-6/STAT3-signaling may thus have contributed to their enhanced production by cultured patients’ pDCs in the earlier study (220).

The reduced IL-6- and IFN-α-induced STAT responses in pDCs remained statistically significant in our study after the exclusion of all patients receiving any treatment for CD (p<0.03, n=3 for the untreated group), suggesting that these findings were not due to medication. 6.3 Monocyte-derived DCs from CD patients have a decreased potential to activate Th17 responses in allogeneic memory cells by soluble mediators In addition to an excessive Th1 activation, gut-related T-cell responses in CD and in animal models of intestinal inflammation are also characterized by an exaggerated activation of IL-17 immunity (16,308,318-321). However, it is not clear to what extent APCs, such as the DCs, contribute to these alterations. Based on animal models, DCs of monocyte origin may be potent APCs to active Th17 cells (217,218). In study IV, we hypothesized that moDCs from CD patients would mount enhanced Th17 responses in allogeneic T cells from non-CD patient donors, demonstrating the direct contribution of APCs to the elevated IL-17 immunity in CD.

In our hands, IL-17-producing cells were not readily induced from the naive CD4+ fraction by any of the implicated cytokine combinations in the presence of polyclonal anti-CD3/CD28 stimulation. Thus, we chose to use the CD4+CD45RO+CD45RA- memory-cell fraction for the comparisons between the moDCs from CD patients and control subjects. To enable the simultaneous testing of all moDC samples with the freshly isolated T cells from the same donors, we used moDC-culture supernatants for the comparisons (which also eliminates the possibility of false positives due to an uneven HLA mismatch, present in an allogeneic-MLR setting). Contrary to the original hypothesis, we found that the upregulation of IL-17A mRNA expression by the LPS-stimulation of moDCs was significantly attenuated in CD when compared with the control subjects (Fig. 11A). This did not seem to be due to CD medication, as the induction of IL-17A transcripts was actually even lower in the patients not receiving any treatment for CD (n=4, see study IV). At the protein level, IL-17A induction was also moderately lower by both LPS- and PGN-stimulated moDCs of patients with CD, but statistical significance was not achieved (Fig. 11B). The LPS-induced mRNA upregulation correlated with the protein-level result in both patients and controls (ρ=0.582, p=0.020 and ρ=0.771, p=0.001, respectively). On the other hand, IL-10 induction in T cells was upregulated at the mRNA level by the LPS stimulation of moDCs from most patients, in contrast to controls (median fold-increase 1.254 vs. 0.963, p=0.031). However, no confirmation could be obtained from the supernatant measurements (see study IV). We did not observe any significant alterations in the expression of RORC, IL-22, IFN-γ, or IL-4 transcripts. When activated in the presence of supernatants from unstimulated DCs, no significant

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differences in T-cell responses (as measured by mRNA transcription or protein secretion) could be detected between the patients and control subjects (see study IV for further information).

Accompanying the attenuated IL-17 induction in memory cells, LPS-activated moDCs from the patients showed decreased transcription of IL-1β and IL-6 when compared with the control subjects (Fig. 12), whereas no differences in mRNA transcript levels for TGF-β1 or IL-12p19 were seen. PGN-induced or spontaneous cytokine expression did not differ between the study groups. No significant differences were detected in the secretion of IL-1β, IL-6, IL-10, IL-12p70, or IL-23 from moDCs (see study IV). Although IL-17A mRNA upregulation in T cells exhibited a strong and highly significant positive correlation with both IL-1β and IL-6

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(at both mRNA and protein level) with PGN stimulation of moDCs (see study IV), we could not detect any significant correlations between the IL-17A mRNA upregulation in T cells and moDC cytokine production in the LPS stimulation of moDCs, with the exception of a trend for a positive correlation between the secreted IL-6 and IL-17A mRNA induction in patients (ρ=0.465, p=0.070). In the light of these results, and the fact that no significant differences in IL-1β, IL-6, or IL-23 could be detected in moDC supernatants between the patients and controls, it seems possible that factors other than these cytokines may have contributed to our finding of altered T cell IL-17A induction.

We also assessed the LPS- and PGN-induced upregulation of the costimulatory molecules CD80 and ICOS ligand by RT-qPCR. The upregulation of CD80 by LPS stimulation was moderately higher in the control subjects than CD patients, approaching statistical significance (p=0.058), whereas no difference in ICOS ligand upregulation was seen (see study IV).

Defects in the mechanisms of autophagy have been suggested to play a role in CD pathogenesis, especially in patients bearing mutated forms of the NOD2 sensor or autophagy protein ATG16L1 (13,290,303). The process of autophagy is now rapidly gaining attention in studies from a broad range of cellular functions including, but by no means restricted to, the processing and MHC class II-mediated presentation of Ags from internalized pathogens by DCs (290). We asked whether autophagy mechanisms could also be affected by the altered reactivity of moDC seen in CD patients in our study. To this end, we assessed the LPS- and PGN-induced upregulation of mRNA transcripts for the LC3 molecule as an mRNA-level surrogate for the induction of autophagy mechanisms (377,378). Fig. 13 demonstrates a dysregulated induction of LC3 transcription upon moDC activation by LPS in CD patients, in comparison with the uniform upregulation seen in the control subjects (LPS-induced fold-increase 1.22 vs. 1.52, p=0.029). This finding in CD patients, not selected by genetic criteria,

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suggests that defective autophagy induction in DCs may play a role in CD that is not restricted to the NOD2 or ATG16L1 mutant patients. Nonetheless, this preliminary result obviously needs to be confirmed by more specific methods, such as the detection of protein level redistribution of LC3 or LC3I–LC3II conversion. Caution in the interpretation is also warranted by the recent demonstration that both experimental and ATG16L1 mutation-related impairment of autophagy in DCs interferes with the normal autophagy-mediated destabilization of the immunological synapse, leading to an increased, rather than decreased, IL-17 secretion from isolated CD45RO+ T-memory cells (379). However, it is not clear how the latter finding relates to the observation that low-level T cell stimulation dramatically favors IL-17- over IFN-γ-responses in human CD4+ T cells (136).

Our results are in some disagreement with a study by Radwan and colleagues, which found increased phenotypic maturation and cytokine (IL-6, IL-12 and IL-23) production in moDCs of CD patient origin when stimulated with LPS, and apparently also simultaneously with TNF (380). In addition to the fact that a combined TNF + LPS stimulation was used, there appears to be other methodological differences to our study: in the study by Radwan et al. moDCs were generated from adherent CD3-negative PBMCs in RPMI 1640 medium in the presence of human serum albumin but in the absence of serum (which is an unusual approach) and neither moDC phenotype nor viability thus achieved was presented. In contrast, our results are in accordance with those of Salucci et al. (381), who found an impaired activation of cytokine production upon S. typhimurium stimulation of moDCs from CD patients bearing the Leu1007insC mutation of NOD2, but also significantly decreased IL-8 response in the

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WT patients, in comparison with the healthy controls. There also appeared to be a trend among the WT patients for reduced TNF, IL-12p70, and IL-10 production. In this study, bacterial-stimulated moDCs from WT patients induced an enhanced Th1 differentiation from naive allogeneic T cells in MLR. In an earlier report, cultured macrophages from CD patients, but not from UC patients, secreted attenuated levels of IL-8 when stimulated with TNF, C5a, or with wound fluid (382). The reduction in LPS-induced secretion did not reach statistical significance. However, the local inflammatory reaction to E. coli was attenuated in vivo. By microarray, patients’ macrophages also showed a decreased upregulation of other inflammatory mediators, such as IL-1β, when stimulated with MDP. None of these findings were related to NOD2 mutations. These results, and ours, somewhat contradict those of Baumgart and colleagues (52), who observed enhanced TNF and IL-8 secretion from LPS-stimulated human primary blood-derived CD1c+ mDCs from patients, together with higher expression of CD40, as discussed earlier. However, according to the latter report, the CD1c+ DC subset from patients already exhibited higher baseline TNF production and significantly increased CD40 expression without the addition LPS. In conclusion, data from the closely related earlier studies are both in partial agreement and in some disagreement with the findings presented here.

In the light of the major genetic association in CD, the NOD2 mutations, the upregulated IL-17 immunity in CD (discussed in chapter 2) is a bit of a paradox. Patients with these variants exhibit attenuated IL-1 and IL-23 production in APCs and induce weak IL-17 responses when stimulated with MDP (138). Explaining this controversy, it has been demonstrated that chronic NOD2-mediated signaling tolerizes innate immune cells, inhibiting subsequent proinflammatory responses mediated not only by the NOD2, but also by the TLR4 pathway (383). Furthermore, cells homozygous for one of the major CD-predisposing NOD2 variants, Leu1007insC, did not develop this tolerance (383). Nevertheless, also from the aspect of immune system, the simplest hypothesis regarding the role of NOD2 mutations in the pathogenesis of CD is that of defective antimicrobial defense, which may lead to an impaired bacterial clearance as a disease-initiating mechanism (15).

Taken together, it appears that the possibility of an innate immunity defect of some kind needs to be given a serious consideration in CD. Moreover, it cannot be excluded that the well-documented enhanced mucosal IL-17 production in patients could be a secondary phenomenon due to a failure in the first-line immune defense. Nevertheless, it seems indisputable that IL-23 plays a role. This role, however, is not necessarily that of adaptive CD4+ Th17 cell upregulation, but may be related to mechanisms within the innate immunity compartment (315,384-386). It has also been demonstrated that the pathogenic adaptive Th17 cells may be driven by IL-23, whereas the non-pathogenic Th17 cells may not (387). Indeed, there are reports questioning the pathogenic role of the Th17 lineage in IBD, suggesting also immunoregulatory activities for IL-17 (388).

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7 Conclusions Our key observations in studies I-IV may be interpreted as follows: I. Enhanced Th17 activation is seen in pediatric T1D, and IL-17A contributes to

the induction of apoptosis in the pancreatic β cells in vitro. However, we did not find signs of an enhanced spontaneous production of the Th17-promoting proinflammatory cytokines IL-1β and IL-6 from APCs of pediatric patients with T1D.

II. Circulating DCs are decreased in children with recent-onset T1D and bear a

phenotypic alteration characterized by a reduced expression of CCR2. The reduction in CCR2 expression does not seem to be related to changes in the systemic levels or autocrine production of CCR2 ligands. In addition, the responsiveness of the NF-κB pathway may be enhanced in children with β-cell autoimmunity. These observations may reflect impaired DC development and predisposition to an altered DC chemotaxis and T-cell activation by the DCs in T1D.

III. The numbers of blood pDCs and BDCA3+ mDCs are decreased in patients

with CD and negatively correlate with the endoscopic disease activity. CD patients may harbor a pDC-restricted IL-6/STAT3 signaling defect, which can be related to the impaired immune regulation in this disease. In addition, the IFN-α-induced signaling response is attenuated in both pDCs and CD1c+ mDCs. CD40 expression is elevated on circulating CD1c+ mDCs from CD patients, in concordance with previous studies, indicating a moderately enhanced activation status of this major mDC subtype.

IV. Monocyte-derived DCs from CD patients have an attenuated ability to

upregulate the IL-17 response in CD4+ memory T cells upon stimulation with bacterial LPS. This is accompanied by a decreased LPS-induced transcription of the Th17-supporting cytokines IL-1β and IL-6 in the DCs. These results imply that a defective innate immune reactivity may be an underlying phenomenon in CD.

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8 Epilogue – general discussion A peculiar feature of this thesis project has been that the exact nature of the DC subsets studied is a matter of debate at a very fundamental level. While it has been addressed during the past decade (as discussed in chapter 2), the precise relationship between the human circulating DCs and DCs in the tissues is certainly not clear at present. Haniffa and colleagues recently pointed out similarities in the transcriptomes between human blood- and tissue-derived mDC subtypes and were also able to find close resemblance to certain DC subsets in mice: BDCA3+ mDCs were related to mouse CD103+ and CD8+ DCs, and CD1c+ mDCs to mouse CD4+ DCs (369). In another recent study performed in unaffected LNs of cancer patients, researchers found human blood mDC equivalents, in addition to pDCs, in all LNs studied (389). They also examined the cell-cycle status of blood-derived DCs and found that >10% of circulating, but not lymphoid tissue, CD1c+ and BDCA3+ mDCs were cycling. These observations led the investigators to propose that the circulating human DC subsets actually represent human LN-resident DCs. The nature of these cells would thus in essence equal to that of the circulating lymphoid-tissue homing DC precursors in mice (32). However, in the light of the results from Haniffa and colleagues, this conclusion may be at least partially incorrect, as the possibility of migration from the peripheral tissues via the afferent lymphatics does not seem to be excluded. Furthermore, circulating mDCs in the human system are potent stimulators of allogeneic T-cells ex vivo (47), while it remains controversial whether (laboratory) mice have circulating DC-like cells capable of robust T-cell activation without further differentiation (61).

We have postulated, also based on the functional properties of these cells, that human circulating DC subsets represent precursors of DCs in the peripheral and/or lymphatic tissues. In addition to their superior availability, the rationale in the investigation of blood-borne cells prior to their translocation to the tissues - such as the intestine, MLNs, the pancreas and its draining LNs - is to make use of the window of less bystander influences due to an inflammatory environment, in order to explore cell-intrinsic alterations that could contribute to the pathogenesis in diseases like T1D and CD (although investigation of immune cells within the disease-relevant tissues bears its evident advantages). Whatever the exact nature of the blood-derived DCs studied in this thesis, certain alterations were observed, and it has been attempted in chapters 5 and 6 to provide a discussion as to their potential functional significance. However, questions also arise regarding the mechanisms by which these deviations are generated.

It is clear that the gut inflammation in CD is accompanied by inflammatory changes that may have a systemic effect, such as the increased levels of IL-6, demonstrated also in our study. However, as exemplified by our failure to find any correlation between the impaired IL-6/STAT3 signaling in CD patients’ pDCs and plasma IL-6, and also between the former and levels of C-reactive protein (data not

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shown), mechanisms related to systemic inflammation do not necessarily play a role. In T1D, there was no correlation between our finding of reduced CCR2 expression and the available data on systemic metabolic/homeostatic disturbances, i.e., blood pH and plasma-glucose concentration at diagnosis. Nonetheless, increased gut permeability occurs not only in CD but also in T1D (13,17), and several factors such as both microbial and endogenous (e.g., calprotectin) PRR-ligands (101), could be released to the circulation and then modulate DC phenotype and function. However, if it is assumed that our findings were not secondary to inflammatory mediators or metabolic disturbances in the circulation, alternative explanations may be considered. Given the probable life cycle of most blood-borne DCs in relation to anatomy (see chapter 2), it appears that the alterations observed by us in the primary blood DCs should then logically be implemented in the level of bone marrow. Some evident possibilities seem to emerge: first, these phenomena could theoretically be genetically determined solely by germline variations, but it is apparent that genetic data as a whole does not support this possibility. Second, analogous to neoplasia, acquired genetic defects might be gathered that predispose patients to DC alterations. However, no clonality is suggested by the rather uniform expression/activation patterns mostly observed in DCs in our study. Third, these phenomena could be brought about by altered epigenetic modification, which could be enforced by some known or yet unidentified disease-predisposing environmental factors. Furthermore, the possibility of infection, e.g., of viral origin, cannot be fully excluded. Last, it seems clear that inflammation-related mechanisms or homeostatic/metabolic disturbances could also operate at the level of the bone marrow. Although technically challenging, and perhaps ethically questionable, it would be informative to study bone marrow DC precursors in human patients.

Regardless of the mechanism, the findings from both T1D and CD in our study – if not secondary in nature - seem to point towards a rather attenuated function of the innate immune system, instead of the more conventional idea of autoimmunity (T1D) or inflammatory disease (CD) as a manifestation of immunological hyperreactivity. Indeed, the idea of CD as primarily a form of immunological deficiency has been presented already in the 1970s (390,391), and increasingly discussed from the viewpoint of innate immunity during the past few years (391,392). In our study, pDCs were decreased in CD patients, whereas the reduction in the major CD1c+ mDC population was not clearly seen. In addition to the aforementioned possible role of pDCs in the induction of immunological tolerance, it may be worth considering in this context also the key role played by pDCs in the antiviral responses. Moreover, besides recognizing MDP, NOD2 has been demonstrated to induce antiviral innate responses by sensing ssRNA (289). Although epidemiological connection to viruses is weak (13), it is intriguing from this perspective that mice with a mutated autophagy-related CD susceptibility gene - Atg16L1 - develop CD-like disease when infected with ssRNA virus MNV (393), and that human intestinal LP CD1c+ DCs were reported to produce IL-23 exclusively when activated with resiquimod, a mimic for viral ssRNA (53). Looking at the GWAS data in relation to

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the possibility of a numeric DC deficiency in CD (as opposed to a mere secondary, inflammation-related decrease), the association to JAK2 (288) is of interest, given that the growth factors IL-3, GM-CSF and G-CSF all signal via this kinase (115). While the nature of the CD-associated JAK2 variant does not seem to be clear at present, it has been reported that some other Th17/IL-23 pathway-related risk alleles in CD lead to hyperresponsiveness: the CD-associated STAT3 allele has been connected to an increased IL-6-induced phosphorylation in granulocytes (304), and the protective minor IL23R allele appears to be a loss-of-function variant (394). Then how are these findings compatible with the concept of CD as a condition with a decreased innate-immune reactivity? It may be conceivable that CD could develop on a background of attenuated innate reactivity when the potential for exaggerated IL-23-driven responses is present; the former could provide sustained stimuli due to an impaired microbial clearance, whereas the latter could then carry out many of the effector functions that ultimately result in CD pathology.

In T1D, there are also some data available to support the idea of the disease process being related to an impaired immune reactivity. Enteroviral infections may be associated with T1D (20), and enteroviral RNA has been found more frequently in the small-intestinal samples from T1D patients than samples from control subjects (395). Furthermore, an attenuated upregulation of Th1-related markers upon enterovirus (coxsackievirus B4) stimulation of PBMCs was reported in CD4+ T cells of T1D patients (396). Decreased production of IL-12p70 has also been demonstrated from patients’ moDCs in LPS stimulation (278). These phenomena could be in line with our finding of reduced CCR2 levels on DCs via the requirement of DC CCR2 expression for proper Th1-responses (366). The demonstrations of reduced DC counts in children with recent-onset disease (see chapter 5.4 and (270)), together with the several papers reporting decreased DC-differentiation from the monocyte precursors in vitro (274-277), constitute another line of observations in favor of the possibility of an innate immune defect in T1D.

Finally, if DC-related alterations are involved in the pathogenesis of autoimmune disease, such as T1D, or inflammatory disease, such as CD, is it plausible that a DC-based strategy could be utilized to prevent or treat the conditions? For both T1D and CD, several such strategies have been tested in animal studies, as discussed in chapter 2. In T1D, a phase I clinical safety trial was also carried out with in vitro differentiated autologous DCs treated with antisense oligonucleotides for costimulatory molecules, with no adverse effects (397). Obviously, the aim to induce T-cell tolerance with such tolerogenic DCs would not need to be connected to a DC abnormality of any kind, and at present, it would be premature to consider a treatment regimen under any hypothesis of DC-related defect. However, using a similar strategy of in vitro manipulation of leukapheresis products, DC-specific influence could be achieved. This would likely be an important aspect. Aiming to enhance, e.g., the responsiveness of IL-6-induced STAT3 signaling could be harmful if not carefully confined to the desired population (305). Anti-IL-6 mAb tocilizumab is actually being tested for human IBD, but may not be beneficial (286), which, on the other hand, is of

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interest considering our pDC-related finding. Similarly, an untargeted attempt to upregulate CCR2 could lead to migratory disturbances, such as an increased tissue translocation of innate cells. Although the probable lifespan of a DC is from some days to maybe some weeks (40), long-lasting effects could possibly be achieved via DC-mediated T-cell modulation, preferably in an Ag-specific manner. Even then, subsequent flux of aberrant DCs from the marrow could hamper the results. These considerations seem to suggest that if DC alterations will turn out to be disease-contributing in nature, their therapeutic targeting may require elaborate methods, being possibly incompatible with any "off-the-shelf" strategy. That said, plain numeric or developmental DC defects have been observed, as discussed above, also by our study. Fairly straightforward administration of DC-lineage boosting growth factors Flt3 ligand and/or G-CSF have shown promising effects in the animal models of T1D and CD (36,38,398), and also in human patients with CD (39). Moreover, it may not be inconceivable that some functional defects might also be rectified by artificially driving enhanced DC hematopoiesis, by virtue of their down-stream nature. Accordingly, these regimens may form the basis for relatively DC-targeted treatments that can possibly be further developed and fine-tuned when more knowledge has been gained.

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9 Acknowledgements First, I would like to thank all the patients, their guardians, and also the control subjects who volunteered to donate their samples for the studies presented here; without your contribution no such research could ever be possible.

The work in this thesis was carried out between 2006 and 2013 first in the Department of Viral Diseases and Immunology at the former National Public Health Institute, then in the Department of Vaccination and Immune Protection at the National Institute of Health and Welfare, and also in the Scientific Laboratory of Children’s Hospital within the Helsinki University Central Hospital. I owe my gratitude to the head of the National Institute of Health and Welfare, Professor Pekka Puska, and to the former and present heads of the departments, Professors Tapani Hovi, Ilkka Julkunen and Terhi Kilpi, as well as to the former head of the Scientific Laboratory, Professor Erkki Savilahti, for providing the excellent research facilities. I am deeply thankful to my supervisor, Professor Outi Vaarala, for all the guidance I have received during this period of scientific work. I also greatly appreciate the trust and level of freedom I have always felt under her supervision. Additionally, the good atmosphere she has been able to create and maintain in the laboratory has been an invaluable asset also for me, personally. I would also like to thank my other supervisor, Professor Mikael Knip. His unmatched enthusiasm towards scientific research, together with his invariably positive and friendly attitude, has been truly encouraging throughout these years. I wish to thank the reviewers of this thesis, Docent Marko Pesu and Docent Arno Hänninen, for the many insightful comments that certainly contributed to improve this text. My warmest appreciation goes to my co-authors for their fruitful collaborations, which have also taught me a lot. In particular, Docent Jukka Vakkila generously offered his expertise regarding both methodological and theoretical questions, which was very helpful especially at the beginning of my DC-related work. Taina Sipponen and Paula Klemetti also kindly contributed their time and knowledge that enabled the cohorts of CD patients to be studied in our laboratory.

In addition to his collaboration, I thank Jarno Honkanen for open-minded scientific attitude that has inspired many intriguing discussions throughout these years. I am indebted to Harri Salo for offering me methodological support in the RT-qPCR-related work. I also want to thank Sinikka Tsupari for her extraordinarily skillful and efficient technical assistance in all of the substudies within this thesis, and I owe gratitude for Anneli Suomela and Leena Saarinen for their expert assistance as well.

My other present and former co-workers in Outi’s lab: Terhi R., Kristiina L., Laura O., Tomi P., Minna T., Emma M., Veera H., Hanne R., Niko K., Mirja N., Linnea H., Monika P., Hanna L., Nina E., Merit M., Julia L., Anna K., Arja V., Elina H., Harri L., Camilla V., Maria K., Tarja A., Kaisa J., Hannele L., Teija J.: thank you

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for you kind help I always received whenever I needed it, and for your contribution to that pleasant atmosphere that has prevailed in the lab for all these years.

People in the Children’s Hospital lab and office: Taina H., Heli S., Anna P., Samppa R., Kirsi S., Markku L-K., Leni J., Iiris O., Berta D., Mevlida K., Sinikka H., Juho H., Jukka R., Tiina N., Sirkku K., Minna K., Elsa V., Laura H., Matti K., Katriina K.: thank you for your aid, and for your splendid company at coffee breaks and at the several great parties.

My sincere gratitude goes to Chris Fogarty for checking the grammar in this thesis, and also for many interesting conversations. Besides their friendship, I thank Sakari for numerous discussions about science and related matters, and Pasi and Klaus for helping me to participate in at least some physical exercise every now and then. I also thank my former co-workers and present friends Jiri and Johanna for sharing with me many memorable moments both in the lab and outside of it.

I am grateful to my parents Anne and Paavo and to my sister Elina for supporting me in my choices in life, and for all the help along the way. I thank my parents in-law, Gitta and Sami for, among many other things, showing love and care to our children, helping out with them when our schedules (especially mine) have been busy. I also thank my dear children Teresa and Ossi - with you guys it is always easy to forget work-related issues and remember what is truly most important for me in life. Finally, my wife Anna, for all the love and enormous support during the almost ten years we have now spent together, there really aren’t words to express my gratitude towards you. This study received financial support from Finska Läkaresällskapet, the Sigrid Juselius Foundation, and from the Finnish Medical Foundation. Helsinki, November 2013 Janne Nieminen

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