Endocr J. 2014;61(6):577-88.

Analysis of the expression of candidate genes for type 1 diabetes susceptibility in T cells.

Hisanaga-Oishi Y, Nishiwaki-Ueda Y, Nojima K, Ueda H.

Department of Molecular Endocrinology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan.

 

ABSTRACT:

Type 1 diabetes is characterized by T-cell-mediated autoimmune destruction of pancreatic β-cells. Currently, approximately 50 type 1 diabetes susceptibility genes or chromosomal regions have been identified. However, the functions of type 1 diabetes susceptibility genes in T cells are elusive. In this study, we evaluated the correlation between type 1 diabetes susceptibility genes and T-cell signaling. The expression levels of 22 candidate type 1 diabetes susceptibility genes in T cells from nonobese diabetic (NOD), control C57BL/6 (B6), and NOD-control F1 hybrid mice were analyzed in response to 2 key immunoregulatory cytokines: interleukin-2 (IL-2) and transforming growth factor β (TGF-β). Exogenous gene expression studies were also performed in EL4 and Jurkat E6.1 T-cell lines. Significant differences in the expression of Clec16a, Dlk1, Il2, Ptpn22, Rnls, and Zac1 (also known as Plagl1) were observed in T cells derived from the 3 strains of mice, and TGF-β differentially influenced the expression of Ctla4, Foxp3, Il2, Ptpn22, Sh2b3, and Zac1. We found that TGF-β induced Zac1 expression in both primary T cells and EL4 cells and that exogenous expression of Zac1 and ZAC1 in T-cell lines altered the expression of Il2 and DLK1, respectively. The results of our study indicate the possibility that additional genetic pathways underlying type 1 diabetes susceptibility, including those involving Clec16a, Dlk1, Rnls, Sh2b3, and Zac1 under IL-2 and TGF-β signaling in T cells, may be shared between human and NOD mice.

PMID: 24705559

SUPPLEMENT:

Type 1 diabetes (T1D) is caused by T cell-mediated autoimmune destruction of insulin-producing pancreatic β-cells. Multiple genetic and environmental factors contribute to disease progression in both human and non-obese diabetic (NOD) mice, an inbred strain of mice that spontaneously develops T1D. The major genetic determinants of T1D in both humans and mice are major histocompatibility complex (MHC) class II molecules, which present antigens to CD4+ T cells. On the other hand, familial clustering of different autoimmune diseases has been frequently reported. The occurrence of common features of autoimmune diseases and the coassociation of multiple autoimmune diseases in the same individual or family supports the notion that there may be common genetic factors that predispose an individual to autoimmunity. We previously reported that a T-cell regulatory gene/cytotoxic T lymphocyte antigen 4 gene (CTLA4) was associated with T1D susceptibility (1). Identification of non-MHC T1D susceptibility genes has shown that several common genetic risk pathways are also shared between human and NOD mice, including the gene encoding CTLA4 (1). Genome-wide association studies (GWAS) have been successful in discovering susceptibility loci in T1D. Currently, there are over 50 non-HLA regions that significantly affect the risk for T1D in both humans and animal models. Pervasive sharing of genetic effects in autoimmune disease has also been shown (2).

Two questions remain elusive: how these genes can determine T1D susceptibility, and what are the functional relationships of these non-MHC genes in T cells. We hypothesized that some combinations of subtle changes of non-MHC susceptibility genes within the T cell might play a role in determining how a T cell responses in the recognition of a weak agonist peptide or a self-peptide displayed by MHC in the pancreatic islets. Therefore, in the present study, as a first step to test this hypothesis and to identify the missing link between non-MHC type 1 diabetes susceptibility genes and T cell regulation, we analyzed the regulation of candidate type 1 diabetes susceptibility genes. Specially, we studied the expressional regulation of these genes in their relationship with two key immunoregulatory cytokines, interleukin (IL)-2 (3) and transforming growth factor-β (TGF-β).

First, we found that NOD T cells grew significantly better when stimulated with anti-CD3/CD28 beads and IL-2 than control mice T cells did, whereas the NOD T cell growth was significantly inhibited in the presence of TGF-β.

Next, 22 genes were selected for analysis. The expression of all 22 genes was detected in T cells from both control and NOD mice T cells (Figure 1). Clec16a, Ctla4, Dlk1, Il2ra, Il2rb, Pparg, Ptpn2, Rnls, Sh2b3, Ubash3a, and Vav3 expression was significantly higher in T cells from NOD mice than in those from control mice, whereas Il2, Ptpn22, and Zac1 expression was significantly lower in T cells from NOD mice than in those from control mice.

The importance of this study: The results of our study indicated the possibility that novel genetic pathways, including those involving Clec16a, Dlk1, Rnls, Sh2b3, and Zac1 under IL-2 and TGF-β signaling in T cells, are also shared between human and NOD mice. We hope that our findings will contribute to furthering our understanding of how type 1 diabetes susceptibility genes coordinately function in the immune system to maintain immune homeostasis.

References

1. Ueda H, Howson JM, Esposito L, Heward J, Snook H, et al. 2003 Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506-511
2. Cotsapas C, Voight BF, Rossin E, Lage K, Neale BM, et al. 2011 Pervasive sharing of genetic effects in autoimmune disease. PLoS Genet 7:e1002254
3. Carbonetto P, Stephens M 2013 Integrated enrichment analysis of variants and pathways in genome-wide association studies indicates central role for IL-2 signaling genes in type 1 diabetes, and cytokine signaling genes in Crohn’s disease. PLoS Genet 9:e1003770
4. Ueda H, Morphew MK, McIntosh JR, Davis MM 2011 CD4+ T-cell synapses involve multiple distinct stages. Proc Natl Acad Sci U S A 108:17099-17104

Acknowledgements: This study was supported by funds from the Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology, Japan and by a Grant-in-Aid for Research Activity Start-up (21890129) from the Japan Society for the Promotion of Science (to Hironori Ueda). Hironori Ueda also received support from the SUMITOMO Life Social Welfare Services Foundation and the Takeda Science Foundation and Novo Nordisk Pharma Insulin Research Award.

Contact:

Hironori Ueda MD, PhD
Associate Professor
Department of Molecular Endocrinology
Osaka University Graduate School of Medicine
2-2, Yamadaoka, Suita, Osaka 565-0871, JAPAN
Phone: +81-6-6879-3825, Fax: +81-6-6879-3829
Email: ueda@endo.med.osaka-u.ac.jp

Figure_1
Fig 1. A graphic representing the 22 genes selected for analysis in this study overlaid on a micrograph of a CD4+ T cell (4). Cells were preserved by high pressure freezing and freeze substitution (http://em-lab.berkeley.edu/EML/index.php). Genes that exhibited statistically significant differences after treatment with TGF-β are shown in yellow.

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