The strongly suppressive regulatory T-cells (Tregs) are bridging central and peripheral tolerance mechanisms through thymic derived and peripherally induced subsets. selleck kinase inhibitor A common description of the Treg phenotype is the concurrent expression of CD4 and high constitutive expression of

the alpha chain of the IL-2 receptor (CD25) [10]. To further identify a regulatory population devoid of activated effector T-cells, a number of markers have been described, most notably absent or low expression of the IL-7 receptor CD127 [11] and [12]. However, pinpointing a pure Treg population is a complex task in humans due to the lack of a specific and unique Treg marker together with the heterogeneity of this population. selleck chemicals The transcription factor forkhead box P3 (FOXP3), found to be expressed in CD4+CD25+ Tregs [13], was also found to be transiently expressed in activated effector T-cells [14] and [15]. FOXP3+ Tregs also constitutively express the inhibitory molecule CTLA-4 [16], while other T-cell subsets express this protein only transiently upon activation. In mouse models completely deprived of Tregs, a variety of autoimmune diseases develop and, likewise, FOXP3 mutations in man cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome [17] and [18]. Furthermore, a variety of diseases and autoimmune states have been associated

with impaired Treg function [10], [17], [18], [19] and [20], while re-introduction of CD4+CD25hi Treg cells into non-obese diabetic (NOD) mice has shown to prevent the development of T1D and to inhibit the production of IFN-γ [21]. In the research field of T1D, where study subjects are often children, blood volumes available for T-cell studies are limited and samples given must be carefully handled and

used to its full extent. Cryopreservation of peripheral blood mononuclear cells (PBMC) in liquid nitrogen allows for batch analysis of samples, but the number of Tregs that can be recovered is limited (∼5–7% of CD4+ T-cells) [22]. In vitro expansion of these cells would therefore be most valuable for biological investigations, and, possibly, for adoptive cell therapies. In the present study, we sought triclocarban to investigate the cryostability of Treg associated markers and further to sort and expand Tregs from cryopreserved PBMC of T1D, high-risk and healthy individuals. The aim was to efficiently expand Tregs and to detect any difference in T-cell number and composition among the studied subjects. For the pre-study, sodium-heparinized venous blood samples were obtained from 16 healthy adults (8 females and 8 males, median age 26.5 years, range 23.5–31). For isolation and expansion, sodium-heparinized venous blood samples were obtained from 9 T1D children (4 females and 5 males, median age 10 years, range 4–14) between 20 days and 10 months after diagnosis (median disease duration 3 months).