Increased attention has been drawn to the important role played by regulatory T-cells (Treg) in immune homoeostasis. However, the small numbers of Tregs make them elusive to study. We investigated the cryostability of Tregs and whether they can be expanded from cryopreserved peripheral blood mononuclear cells (PBMCs). Further, to elucidate if there is a difference in ex-vivo frequency or in vitro expansion of Tregs among T1D children (n=9), high-risk (n=7) and healthy (n=10) individuals, Tregs defined as CD4⁺CD25⁺CD127^lo/− were isolated from cryopreserved PBMCs.

Cryopreserved PBMCs maintained a stable expression of Treg-markers. Tregs were efficiently expanded in vitro from all donors and Tregs from T1D children acquired higher FOXP3 expression compared to healthy subjects. T1D children had a significantly lower percentage of Tregs among CD4⁺ T-cells and also lower Treg to CD4⁺CD25⁻ cell ratios compared to healthy individuals.