CD28 Superagonist D665-mediated activation of mouse regulatory T cells maintains their phenotype without loss of suppressive quality.

Regulatory T cells (Tregs) maintain immune homeostasis by regulating the activation of other immune cells. Preclinical studies show that the infusion of Tregs can promote immunological tolerance to allografts and prevent or cure multiple autoimmune diseases. However, Treg therapy is limited by high numbers of cells required to induce tolerance. In this study, we aimed at improving the in vitro expansion of sort purified mouse Tregs using the CD28 Superagonist (CD28-SA) D665 and comparing it to the conventional expansion using anti-CD3/anti-CD28 Dynabeads®. CD28-SA-stimulated Tregs expanded more than Dynabead®-stimulated Tregs while maintaining their phenotype by expressing the same level of CD4, CD25 and Foxp3. CD28-SA-expanded Tregs produced comparable amounts of IL-10 and TGFβ while showing a slightly superior suppressive capacity compared to Dynabead®-stimulated Tregs. Thus, stimulating murine Tregs with the CD28-SA is a promising alternative since it maintains their suppressive capacity without altering their phenotype and yields a higher fold expansion within 14 days.