At-risk and recent-onset type 1 diabetic subjects have increased apoptosis in the CD4+CD25+ T-cell fraction.

BACKGROUND: In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ (FoxP3+) suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+(high) T cells in human disease. In this study we measure apoptosis of CD4+CD25+(high) T cells to see if it could contribute to reduced suppressive activity of these cells. METHODS AND FINDINGS: T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+(high) and CD4+CD25- T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+(high) T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2-3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+(high) T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+(high) T cells committed to apoptosis at a higher percentage 15.3+/-2.2 compared to FoxP3+ve CD4+CD25+(high) T cells in control subjects 6.1+/-1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+(high) T cells p = 0.0007 and p = 0.007, respectively. CONCLUSIONS: There is a higher level of ongoing apoptosis in CD4+CD25+(high) T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+(high) T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects.

Difference in gene expression profiles between human CD4+CD25+ and CD4+CD25- T cells.

We studied the gene expression profiles of human CD4+CD25+ and CD4+CD25- T cells by using cDNA microarrays. Our preliminary results indicate that there are likely significant differences in the regulation of apoptosis, cell cycle, cytokine receptor, cell-cell interaction, and stress pathway genes between these two subtypes of T cells.

The design of a gene chip for functional immunological studies on a high-quality control platform.

We have created an immunology-related microarray chip containing primarily known genes with well-studied functional properties. By looking at known genes rather than expressed sequence tags, we hope to gain a better understanding of immunological pathways and how they work. The immunology gene chip contains genes from the following functional categories: T cell genes; B cell genes; dendritic cell genes; chemokine and cytokine genes; apoptosis genes; cell cycle genes; cell interaction genes; general hematology and immunology genes; and adhesion genes. We have also developed a novel three-color cDNA array platform in which arrays are directly visualized before hybridization, which allows us to select only high-quality chips for our experiments. In an effort to provide quantitative quality control for each array element as well as the entire chip, we have developed Matarray, a software package for image processing and data acquisition. With Matarray, we have built a quantitative data filtering and normalization scheme that has proved to be more efficient than the existing methods. The list of immunology chip genes is available from the authors.