Islet antigen-specific T cells and regulatory T cells in type 1 diabetes

T cells are the key players in the development of type 1 diabetes (T1D), mediating autoimmune reactions leading to the destruction of insulin producing beta cells in the islets. We aimed to analyze the role of different T-cell subtypes in the autoimmunity and pathogenesis of T1D. The frequency of islet antigen-specific (GAD65-, proinsulin-, and insulin-specific) CD4+ T cells was investigated in vitro in T1D patients, at-risk individuals (diabetes-associated autoantibody positive), and in controls, using MHC class II tetramers. An overall higher frequency of CD4+ T-cells recognizing the GAD65 555−567 peptide was detected in at-risk individuals. In addition, increased CD4+ T-cell responses to the same GAD65 epitope displaying a memory phenotype were observed in at-risk and diabetic children, which demonstrate a previous encounter with the antigen in vivo. Avidity and phenotypic differences were also observed among CD4+ T-cell clones induced by distinct doses of GAD65 autoantigen. T-cell clones generated at the lowest peptide dose displayed the highest avidity and expressed more frequently the TCR Vβ5.1 chain than low-avidity T cells. These findings raise attention to the antigen dose when investigating the diversity of antigen-specific T cells. Furthermore, an increased regulatory response during the preclinical phase of T1D was also found in genetically at-risk children. Higher frequencies of regulatory T (Treg) cells (CD4+CD25_high HLA-DR-/CD69-) and natural killer T (NKT) cells (CD161+Vbeta11+) were observed in children with multiple autoantibodies compared to autoantibody-negative controls. Taken together, these data showed increased frequency of islet-specific CD4+ T-cells, especially to the GAD65 555-567 epitope, and Treg and NKT cell upregulation in children at-risk for T1D, suggesting their importance in T1D pathogenesis

Modulation of Signaling Molecules by Human Leucocyte Antigen B27; Special Reference to STAT1

Reactive arthritis (ReA) is an inflammatory joint disease, which belongs to the group of Spondyloarthritis (SpA). It may occur after infections with certain gram-negative bacteria such as Salmonella and Yersinia. SpAs are strongly associated with the human leucocyte antigen (HLA)-B27. Despite active research, the mechanism by which HLA-B27 causes disease susceptibility is still unknown. However, HLA-B27 has a tendency to misfold during assembly. It is possible that the misfolding of HLA-B27 could alter signaling pathways and/or molecules involved in inflammatory response in cells. We have earlier discovered that in HLA-B27-positive cells the interaction between the host and causative bacteria is disturbed. Our recent studies indicate that the expression of HLA-B27 may alter certain signaling molecules by disturbing their activation. The aim of this study was to investigate whether the expression of HLA-B27 disturbs the signaling molecules, especially the phosphorylation of transcription factor STAT1. STAT1 is an important mediator of inflammatory responses. Our results show that the phosphorylation of the STAT1 is significantly altered in HLA-B27-expressing U937 monocytic cells compared with control cells. STAT1 tyrosine 701 is more strongly phosphorylated in HLAB27- expressing cells; whereas the phosphorylation of STAT1 serine 727 is prolonged. Phosphorylation of STAT1 was discovered to be dependent on protein kinase PKR. Furthermore, we found out that the expression of posttranscriptional gene regulator HuR was altered in HLA-B27-expressing cells. We also detected that HLA-B27-positive cells secrete more interleukin 6, which is an important mediator of inflammation. These results help to understand how HLA-B27 may confer susceptibility to SpAs.

The expression of HLA-B27 modulates intracellular signaling in human monocytic macrophages

Reactive arthritis (ReA) is an inflammatory joint disease triggered by certain bacterial infections e.g. gastroenteritis caused by Salmonella. ReA is strongly associated to HLA-B27. However, the mechanism behind this association is unknown but it is suggested that the bacteria or bacterial compartments persist in the body. In this study, it was investigated whether the intracellular signaling is altered in HLA-B27- transfected U937 monocytic macrophages. Moreover, the contribution of HLA–B27 heavy chain (HC) misfolding was of interest. The study revealed that p38 activity plays a crucial role in controlling intracellular Salmonella Enteritidis in U937 cells. The replication of intracellular bacteria was dependent on p38 kinase and the activity of p38 was dysregulated in HLA-B27- transfected cells expressing misfolding heavy chains (HCs). Also the double-stranded RNA -dependent kinase (PKR) that modifies p38 signaling was overexpressed and hypophosphorylated upon infection and lipopolysaccharide stimulation. The expression of CCAAT enhancer binding protein beta (C/EBPβ) was found to be increased after infection and stimulation. Increased amount of full length human antigen R (HuR), disturbed HuR cleavage and reduced dependence on PKR after infection were observed. All the findings were linked to HLA-B27 HCs containing misfoldingassociated glutamic acid 45 (Glu45) at the peptide binding groove. The results indicate that the expression of HLA-B27 modulates the intracellular environment of U937 monocytic macrophages by altering signaling. This phenomenon is at least partially associated to the HLA-B27 misfolding. These observations offer a novel explanation how HLA-B27 may modulate inflammatory response induced by ReA-triggering bacteria.

Genes outside The Hla Region affecting Susceptibility to Type 1 Diabetes - The Role of Iddm2 and Iddm9 in the Finnish Population

Tyypin 1 diabeteksen perinnöllinen alttius Suomessa - HLA-alueen ulkopuolisten alttiuslokusten IDDM2 ja IDDM9 rooli taudin periytymisessä HLA-alue, joka sijaitsee kromosomissa 6p21.3, vastaa noin puolesta perinnöllisestä alttiudesta sairastua tyypin 1 diabetekseen. Myös HLA-alueen ulkopuolisten lokusten on todettu liittyvän sairausalttiuteen. Näistä kolmen lokuksen on varmistettu olevan todellisia alttiuslokuksia ja lisäksi useiden muiden, vielä varmistamattomien lokusten, on todettu liittyvän sairausalttiuteen. Tässä tutkimuksessa 12:n HLA-alueen ulkopuolisen alttiuslokuksen kytkentä tyypin 1 diabetekseen tutkittiin käyttäen 107:aa suomalaista multiplex-perhettä. Jatkotutkimuksessa analysoitiin IDDM9-alueen kytkentä ja assosiaatio sairauteen laajennetuissa perhemateriaaleissa sekä IDDM2-alueen mahdollinen interaktio HLA-alueen kanssa sairauden muodostumisessa. Lisäksi suoritettiin IDDM2-alueen suojaavien haplotyyppien alatyypitys tarkoituksena tutkia eri haplotyyppien käyttökelpoisuutta sairastumisriskin tarkempaa ennustamista varten. Ensimmäisessä kytkentätutkimuksessa ei löytynyt koko genomin tasolla merkitsevää tai viitteellistä kytkentää tutkituista HLA-alueen ulkopuolisista lokuksista. Voimakkain havaittu nimellisen merkitsevyyden tavoittava kytkentä nähtiin IDDM9-alueen markkerilla D3S3576 (MLS=1.05). Tutkimuksessa ei kyetty varmistamaan tai sulkemaan pois aiempia kytkentähavaintoja tutkituilla lokuksilla, mutta IDDM9-alueen jatkotutkimuksessa havaittu voimakas kytkentä (MLS=3.4) ja merkitsevä assosiaatio (TDT p=0.0002) viittaa vahvasti siihen, että 3q21-alueella sijaitsee todellinen tyypin 1 diabeteksen alttiusgeeni, jolloin alueen kattava assosiaatiotutkimus olisi perusteltu jatkotoimenpide. Sairauteen altistava IDDM2-alueen MspI-2221 genotyyppi CC oli nimellisesti yleisempi matalan tai kohtalaisen HLA-sairastumisriskin diabeetikoilla, verrattuna korkean HLA-riskin potilaisiin (p=0.05). Myös genotyyppijakauman vertailu osoitti merkitsevää eroa ryhmien välillä (p=0.01). VNTR-haplotyyppitutkimus osoitti, että IIIA/IIIA-homotsygootin sairaudelta suojaava vaikutus on merkitsevästi voimakkaampi kuin muiden luokka III:n genotyypeillä. Nämä tulokset viittaavat IDDM2-HLA -vuorovaikutukseen sekä siihen että IDDM2-alueen haplotyyppien välillä esiintyy etiologista heterogeniaa. Tämän johdosta IDDM2-alueen haplotyyppien tarkempi määrittäminen voisi tehostaa tyypin 1 diabeteksen riskiarviointia.

Homogeneous Assays for Simplified Screening of HLA-conferred Genetic Disease Risk

The increasing incidence of type 1 diabetes has led researchers on a quest to find the reason behind this phenomenon. The rate of increase is too great to be caused simply by changes in the genetic component, and many environmental factors are under investigation for their possible contribution. These studies require, however, the participation of those individuals most likely to develop the disease, and the approach chosen by many is to screen vast populations to find persons with increased genetic risk factors. The participating individuals are then followed for signs of disease development, and their exposure to suspected environmental factors is studied. The main purpose of this study was to find a suitable tool for easy and inexpensive screening of certain genetic risk markers for type 1 diabetes. The method should be applicable to using whole blood dried on sample collection cards as sample material, since the shipping and storage of samples in this format is preferred. However, the screening of vast sample libraries of extracted genomic DNA should also be possible, if such a need should arise, for example, when studying the effect of newly discovered genetic risk markers. The method developed in this study is based on homogeneous assay chemistry and an asymmetrical polymerase chain reaction (PCR). The generated singlestranded PCR product is probed by lanthanide-labelled, LNA (locked nucleic acid)-spiked, short oligonucleotides with exact complementary sequences. In the case of a perfect match, the probe is hybridised to the product. However, if even a single nucleotide difference occurs, the probe is bound instead of the PCR product to a complementary quencher-oligonucleotide labelled with a dabcyl-moiety, causing the signal of the lanthanide label to be quenched. The method was applied to the screening of the well-known type 1 diabetes risk alleles of the HLA-DQB1 gene. The method was shown to be suitable as an initial screening step including thousands of samples in the scheme used in the TEDDY (The Environmental Determinants of Diabetes in the Young) study to identify those individuals at increased genetic risk. The method was further developed into dry-reagent form to allow an even simpler approach to screening. The reagents needed in the assay were in dry format in the reaction vessel, and performing the assay required only the addition of the sample and, if necessary, water to rehydrate the reagents. This allows the assay to be successfully executed even by a person with minimal laboratory experience.

Free Prostate-specific Antigen Forms and Kallikrein-related Peptidase 2: Tools for Prostate Cancer Diagnostics

Prostate-specific antigen (PSA) is a marker that is commonly used in estimating prostate cancer risk. Prostate cancer is usually a slowly progressing disease, which might not cause any symptoms whatsoever. Nevertheless, some cases of cancer are aggressive and need to be treated before they become life-threatening. However, the blood PSA concentration may rise also in benign prostate diseases and using a single total PSA (tPSA) measurement to guide the decision on further examinations leads to many unnecessary biopsies, over-detection, and overtreatment of indolent cancers which would not require treatment. Therefore, there is a need for markers that would better separate cancer from benign disorders, and would also predict cancer aggressiveness. The aim of this study was to evaluate whether intact and nicked forms of free PSA (fPSA-I and fPSA-N) or human kallikrein-related peptidase 2 (hK2) could serve as new tools in estimating prostate cancer risk. First, the immunoassays for fPSA-I and free and total hK2 were optimized so that they would be less prone to assay interference caused by interfering factors present in some blood samples. The optimized assays were shown to work well and were used to study the marker concentrations in the clinical sample panels. The marker levels were measured from preoperative blood samples of prostate cancer patients scheduled for radical prostatectomy. The association of the markers with the cancer stage and grade was studied. It was found that among all tested markers and their combinations especially the ratio of fPSA-N to tPSA and ratio of free PSA (fPSA) to tPSA were associated with both cancer stage and grade. They might be useful in predicting the cancer aggressiveness, but further follow-up studies are necessary to fully evaluate the significance of the markers in this clinical setting. The markers tPSA, fPSA, fPSA-I and hK2 were combined in a statistical model which was previously shown to be able to reduce unnecessary biopsies when applied to large screening cohorts of men with elevated tPSA. The discriminative accuracy of this model was compared to models based on established clinical predictors in reference to biopsy outcome. The kallikrein model and the calculated fPSA-N concentrations (fPSA minus fPSA-I) correlated with the prostate volume and the model, when compared to the clinical models, predicted prostate cancer in biopsy equally well. Hence, the measurement of kallikreins in a blood sample could be used to replace the volume measurement which is time-consuming, needs instrumentation and skilled personnel and is an uncomfortable procedure. Overall, the model could simplify the estimation of prostate cancer risk. Finally, as the fPSA-N seems to be an interesting new marker, a direct immunoassay for measuring fPSA-N concentrations was developed. The analytical performance was acceptable, but the rather complicated assay protocol needs to be improved until it can be used for measuring large sample panels.

The Roots of Neurofibromas in Neurofibromatosis 1 — A Question of Multipotency, Differentiation and Hiding

Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome that affects about 1 in 3500 individuals worldwide. NF1 is caused by mutations in the NF1 gene that encodes the tumor suppressor protein neurofibromin, an inactivator of the Ras oncogene. The hallmarks of NF1 include pigmentary lesions of the skin, Lisch nodules of the iris and cutaneous neurofibromas. Cutaneous neurofibromas are benign tumors composed of all the cell types of normal peripheral nerve. The traditional view of neurofibroma development has been that cutaneous neurofibromas arise from the disruption of the small nerve tributaries of the skin and subsequent proliferation of the resident cells. The second hit mutation in the NF1 gene has been considered as a prerequisite for neurofibroma development. The second hit is detectable in a subpopulation of primary Schwann cells cultured from neurofibromas. This thesis challenges the traditional concept of neurofibroma development. The results show that cutaneous neurofibromas are intimately associated with hair follicular structures and contain multipotent precursor cells (NFPs), suggesting that neurofibromas may arise from the multipotent cells which reside in hair follicles. Furthermore, this study presents that neurofibroma-derived Schwann cells that harbor bi-allelic inactivation in the NF1 gene express HLA class II genes and may act as nonprofessional antigen presenting cells. The CD4- and FoxP3-positive cells detected in cutaneous neurofibromas suggest that these cells may represent regulatory T cells (Tregs) which interact with HLA II –positive cells and aid the tumor cells in hiding from the immune system and are thus mediators of immune tolerance. This thesis also investigated neurofibroma development in the oral cavity and the use of different biomarkers to characterize cellular differentiation in neurofibromas. The results revealed that oral neurofibromas are not rare, but they usually appear as solitary lesions contrary to multiple cutaneous neurofibromas and present high heterogeneity within and between tumors. The use of class III beta-tubulin as a marker for neuronal differentiation led to an unexpected finding showing that multiple cell types express class III beta-tubulin during mitosis. The increased understanding of the multipotency of tumor cells, cellular differentiation and ability to hide from immune system will aid in the development of future treatments. Specifically, targeting Tregs in NF1 patients could provide a novel therapeutic approach to interfere with the development of neurofibromas.