Comparative analysis of human and mouse transcriptomes of Th17 cell priming.

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models.

Standard of hygiene and immune adaptation in newborn infants.

The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu.

Autoantigen-specific memory CD4+ T cells are prevalent early in progression to Type 1 diabetes.

Autoreactive CD4(+) T cells contribute to the destruction of insulin producing beta cells in Type 1 diabetes (T1D). Using MHC class II tetramers, we have analyzed the frequency of GAD65- (274-286; 555-567) and insulin- (A1-15; A6-21) specific CD4(+) T cells in 31 children with T1D, 65 multiple autoantibody-positive children and 93 HLA- and age-matched controls. In a smaller group of children T-cell responses of memory origin to the same autoantigens were investigated. We observed a higher response to GAD65 555-567 in the autoantibody-positive children than in the controls (P=0.017). Memory T-cell responses to GAD65 555-567 were more frequent among T1D patients (P=0.025) and autoantibody-positive (P=0.054), while all controls were negative (n=28). In summary, the presence of antigen experienced GAD65-specific T cells in the subjects with diabetes-associated autoimmunity is encouraging for further directions in the prediction of T1D.

A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: a study of human autoreactive CD4+T cells specific for GAD65.

T cells specific for pancreatic islet proteins can be detected in type 1 diabetes patients and at-risk individuals, suggesting a failure of the central tolerance and negative selection. We addressed the question, how antigen dose shapes the diversity of CD4+ autoreactive T cells specific for glutamate decarboxylase 65 (GAD65) in a healthy HLA-DR*0404+ individual, with a persistent GAD65-specific T-cell response. CD4+T cells from this subject were stimulated with decreasing concentrations of the GAD65 555-567 (557I) peptide, and T-cell clones were derived from the tetramer-binding cell population. Functional and structural avidity, TcR-Vß usage, and cytokine profiles were investigated at a clonal level. T-cell clones established with a low antigen dose (0.1 and 1 µg/ml) displayed higher avidity in contrast to the clones established with the highest antigen dose (10 µg/ml; Mann-Whitney U test, p = 0.003 and 0.006, respectively). The T-cell clones stimulated with the lowest peptide dose also had a higher tetramer-binding affinity than clones stimulated with the highest dose (p = 0.026). The majority (60.0%) of the high-avidity clones expressed TcR-Vß5.1 chain whereas only one (12.5%) low-avidity clone did. All clones displayed Th0/Th2 cytokine profiles, but intermediate and high-avidity clones produced more IL-10 than low-avidity clones (p = 0.032). The results demonstrate an important role of the antigen dose in the determination of characteristics of the responding T-cell repertoire. High IL-13 and IL-10 production by GAD65-reactive T cells suggests a more anti-inflammatory profile of this healthy individual underlying protection from T1D.

Reduced CD4+T cell activation in children with type 1 diabetes carrying the PTPN22/Lyp 620Trp variant.

The 620Trp variant of the LYP protein, encoded by the lymphoid tyrosine phosphatase 22 gene (PTPN22), is associated with autoimmunity. In this study we aimed at characterising the role of this variant on lymphocyte activation. We analysed cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) and CD4(+)T cells in a cohort of clinically non-diabetic, multiple autoantibody-positive children, healthy controls and in children with type 1 diabetes (T1D). We found a decreased proliferation and IL-2 production of CD4(+)T cells after anti-CD3/anti-CD28 stimulation (p=0.04 for IL-2) among T1D patients. In addition, a profoundly decreased intracellular calcium flux in CD4(+)T cells after PHA stimulus was detected among 620Trp carriers. In contrast, no effect of this polymorphism on tuberculin and tetanus toxoid induced PBMC proliferation and cytokine secretion was observed in autoantibody positive children, healthy controls and children with newly-diagnosed T1D. In conclusion, the LYP 620Trp variant is associated with reduced activation, proliferation and IL-2 production in CD4(+)T cells among T1D patients. In accordance with our previous findings on the key role of this variant on disease progression, this mechanism is likely to contribute to the development of beta-cell specific autoimmunity.

Circulating CD4+CD25 high regulatory T cells and natural killer T cells in children with newly diagnosed type 1 diabetes or with diabetes-associated autoantibodies.

Type 1 diabetes is a T-cell-mediated autoimmune disease in which insufficient regulatory mechanisms are perceived to be involved in the pathogenesis. We used flow cytometry to analyze the proportion of CD4(+)CD25(high) regulatory T cells and natural killer T (NKT) cells in peripheral blood obtained from 25 children with newly diagnosed type 1 diabetes, 21 nondiabetic subjects positive for two or more diabetes-associated autoantibodies, and from 39 autoantibody-negative age- and HLA-matched control subjects. CD4(+)CD25(high) T cells were also stained for additional markers HLA-DR, CD69, and CD62L. As NKT cell markers, we used CD161, V beta 11, and V alpha 24. The frequency of CD4(+)CD25(high)HLA-DR(-) T cells was significantly higher in multiple autoantibody-positive children than in controls (P = 0.021). We also detected a significantly higher level of CD4(+)CD25(high)HLA-DR(-) and CD4(+)CD25(high)CD69(-) T cells among children expressing three to four autoantibodies when compared to the controls (P = 0.004 and P = 0.048, respectively). The proportions of CD161(+)V beta 11(+) or V alpha 24(+)V beta 11(+) NKT cells were similar in all three groups of children studied. Interestingly, children with only two autoantibodies had a higher level of CD161(+)V beta 11(+) NKT cells than the controls (P = 0.002). Our data might be interpreted as indicative of an intensified regulatory response of regulatory T cells and NKT cells during the preclinical phase of the disease.

GAD65- and proinsulin-specific CD4+ T-cells detected by MHC class II tetramers in peripheral blood of type 1 diabetes patients and at-risk subjects.

In type 1 diabetes the major loss of insulin producing beta-cells is caused by autoreactive T-cells specific for antigens expressed by the pancreatic islets. In this study we have analyzed the prevalence of glutamate decarboxylase 65 (GAD65)- and proinsulin-specific CD4(+) T-cells in type 1 diabetes patients, at-risk subjects and in HLA-matched control children. Peripheral blood mononuclear cells were cultured in the presence of two different GAD65 peptides (555-567, 557I and 274-286) or with a proinsulin (B24-C36) peptide for 10-11days. The autoreactive T-cells were detected using antigen specific-MHC class II tetramers by flow cytometry. Our results show that 11 of 18 (61%) type 1 diabetes patients and 7 of the 20 (35%) at-risk subjects were positive for one of the three GAD65 or proinsulin-containing tetramers, whereas only 2 of 21 (9.5%) controls had tetramer binding cells (p = 0.0007 type 1 diabetes vs. controls and p = 0.0488 at-risk subjects vs. controls, Chi-square test). Type 1 diabetes patients responded to all three peptides. At-risk subjects recognized also the GAD65 555-567 557I peptide, while none of the controls responded to it. In conclusion, type 1 diabetes patients and at-risk subjects have a significantly higher prevalence of GAD65- and proinsulin-specific CD4(+) T-cells than the control subjects.