Abstract
BackgroundIn 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 FindingsT-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.ConclusionsThere 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.
Highlights
Type 1 diabetes (T1D) is an autoimmune disorder in which autoreactive T cells attack pancreatic b-cells, leading to a lifelong dependency on insulin
In all clinical groups studied, two T-cell subsets (CD4+CD252 and CD4+CD25+high) were collected and analyzed ex vivo for 1. proliferation/suppression; 2. apoptosis determination using nucleic acid stains (YOPRO1/7AAD); 3. intracellular staining for FoxP3 and 4. intracellular staining for aaCas3
The CD4+CD252 T cells are used as responders in a suppression assay and they served as an internal control for the measurement of apoptosis
Summary
Type 1 diabetes (T1D) is an autoimmune disorder in which autoreactive T cells attack pancreatic b-cells, leading to a lifelong dependency on insulin. Most studies in murine T1D models have found that autoreactive T cells are resistant to apoptosis[3] This predisposes an individual to autoimmune disease as autoreactive T cells find their way to the periphery and attack the target organ. 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 (15.362.2) compared to FoxP3+ve CD4+CD25+high T cells in control subjects (6.161.7) (p,0.002). 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). 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
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