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

Interaction between Treg Apoptosis Pathways, Treg Function and HLA Risk Evolves during Type 1 Diabetes Pathogenesis

We have previously reported increased apoptosis of regulatory T cells (Tregs) in recent-onset Type 1 Diabetes subjects (RO T1D) in the honeymoon phase and in multiple autoantibody-positive (Ab+) subjects, some of which are developing T1D. We have also reported that increased Treg apoptosis was associated with High HLA risk and that it subsided with cessation of honeymoon period. In this report, we present results generated using genetics, genomics, functional cell-based assays and flow cytometry to assess cellular changes at the T-cell level during T1D pathogenesis. We measured ex vivo Treg apoptosis and Treg function, surface markers expression, expression of HLA class II genes, the influence of HLA risk on Treg apoptosis and function, and evaluated contribution of genes reported to be involved in the apoptosis process. This integrated comprehensive approach uncovered important information that can serve as a basis for future studies aimed to modulate Treg cell responsiveness to apoptotic signals in autoimmunity. For example, T1D will progress in those subjects where increased Treg apoptosis is accompanied with decreased Treg function. Furthermore, Tregs from High HLA risk healthy controls had increased Treg apoptosis levels and overexpressed FADD but not Fas/FasL. Tregs from RO T1D subjects in the honeymoon phase were primarily dying through withdrawal of growth hormones with contribution of oxidative stress, mitochondrial apoptotic pathways, and employment of TNF-receptor family members. Ab+ subjects, however, expressed high inflammation level, which probably contributed to Treg apoptosis, although other apoptotic pathways were als

Differential gene expression in the three subject groups relative to Low HLA risk group.

<p>Ratio of gene expression between Tregs and CD25− from each subject was first obtained and averaged for each group before comparison to the Low HLA risk control subject group. A) fold change genes expression in the High HLA risk healthy control (n = 8), Ab+ subjects (n = 4), RO T1D subjects (n = 4), relative to Low HLA risk subject group (n = 5), B) fold change of genes uniquely expressed in RO T1D (red bars), Ab+ subjects (dark grey bars) and High HLA risk subjects (light grey bars) relative to Low HLA risk group. C) Genes downregulated in High HLA risk group compared to Low HLA risk group. Cutoff value for presentation of overexpressed genes was >5-fold. * - anti-apoptotic genes.</p

Changes in correlation of HLA risk with Treg apoptosis and Treg function in subject cohorts representing stages of T1D development.

<p>In High HLA risk both healthy control and LS T1D subjects, increased Treg apoptosis significantly correlated with increased Treg function (p = 0.017 and p = 0.042, respectively). High HLA risk RO T1D subjects showed opposite correlation: increased Treg apoptosis was correlated with decreased Treg function (p = 0.027). Multiple Ab+ subjects did not show correlation between Treg apoptosis and function in High HLA risk group (p = 0.95). Although some trends were present, Low HLA risk groups did not show correlation with Treg apoptosis and function.</p

Demographic data of studied population.

<p>All data presented as (mean value)±SE; N/A – not available;</p>*<p>- not applicable.</p

Fas/FasL T-cell apoptosis prevalent in RO T1D, LS T1D and Low HLA risk controls.

<p>A) <i>In vitro</i> soluble FasL caused significantly lower T cell apoptosis (in both naïve-CD25 and Tregs) in healthy High HLA risk control subjects compared to RO T1D T cells (for both cell subset comparisons Mann-U-Whitney test was p = 0.016), suggesting that Fas/FasL is one of the prevalent apoptosis mechanism in RO T1D subjects, but not in High HLA risk control subjects. B) Treg apoptosis caused by soluble FasL (1/40 dilution – 600 ng/ml) in healthy control, RO T1D and LS T1D subjects was significantly reduced with pretreatment with caspase 3 inhibitor Z-DEVD in majority of subjects. However, analysis of RO T1D subjects alone (red symbols) showed their better responsiveness to pretreatment with Ac-IETD compared to Z-DEVD. C) Activation-induced Treg apoptosis by anti-CD3 was moderately prevented through treatment of both inhibitors, suggesting an involvement of both caspase 3 and 8 in this apoptosis pathway in all subject groups. RO T1D subjects were labeled with red symbols. Mann-U-Whitney test was used for comparisons.</p

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