At-Risk and Recent-Onset Type 1 Diabetic Subjects Have Increased Apoptosis in the CD4+CD25+(high) 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

Apoptosis of CD4+CD25high T Cells in Type 1 Diabetes May Be Partially Mediated by IL-2 Deprivation

from T1D subjects. in T1D may be caught up in a relatively deficient cytokine milieu. function in subjects predisposed to T1D

The type of responder T-cell has a significant impact in a human in vitro suppression assay.

In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4(+)CD25(+high), or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4(+)CD25(low) T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.We investigated human CD4(+)CD25(low) T cells and compared them to CD4(+)CD25(-) T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4(+)CD25(low) T cells divided more rapidly than CD4(+)CD25(-) T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25(low) compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25(low) T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively).The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases

Figure 5

<p>CD4+CD25+^high T cells suppressive potential across major clinical groups. Control subjects show statistically higher suppression compared to the other three groups recent-onset T1D, high at-risk and longstanding T1D subjects p = 0.0007, p = 0.007 and p = 0.04, respectively. Standardized suppression assay was performed with 1×10⁴ responder T cells, 10⁴ irradiated PBMCs 5000rad alone and in co-culture with CD4+CD25+^high T cells in 1∶10 ratio, stimulated with aCD3 coated beads 1ug/ml, 3 beads/per cell.</p

Figure 3

<p>Comparison of apoptosis measurement in the same subjects using two different methods. a CD4+CD25+^high T cells apoptosis in three clinical groups using YOPRO/7AAD stain combination; control subjects CD4+CD25+^high T cells apoptosis significantly differs from both recent-onset T1D and high at-risk subjects p = 0.0001 and p = 0.01, respectively. Apoptotic cells are presented on y-axis as YOPRO1+ve. b CD4+CD25+^high T cells apoptosis in the same subjects using intracellular staining of aaCas3. CD4+CD25+^high T cells apoptosis from control subjects significantly differs from both recent-onset T1D and high at-risk subjects p = 0.0006 and p = 0.009, respectively. Spearman's rank correlation coefficient between two apoptosis measurement methods in control and recent-onset T1D subjects is significant r = 0.59 and p = 0.0006. Apoptotic cells are presented on y-axis as aaCas3+ve.</p

Figure 1

<p>Representative FACS pictures of CD4+CD25− and CD4+CD25+^high T cells using YOPRO1/7AAD method from representative subjects of clinical groups. Apoptosis was calculated as the percentage of apoptotic cells YOPRO1+ve/7AAD-ve amongst live cells total 7AAD-ve cells comprising both YOPRO1+ve and YOPRO1-ve cells. This is simply designated as YOPRO1+ve.</p