Determination of optimal mode and strength of secondary polyclonal Treg stimulation.

<p>(A) Treg were primed with anti-CD3+ anti-CD28 microbeads and restimulated with indicated ratios of anti-CD3+ anti-CD28 microbeads or with indicated concentrations of platebound anti-CD3+ soluble anti-CD28 in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [³H]Thymidine incorporation at day 3. (B) Indexed cell yield after secondary polyclonal Treg culture using optimal strength of anti-CD3+ anti-CD28 microbeads (set to 100%) or platebound anti-CD3+ soluble anti-CD28 stimulation. (C) Cell division pattern and survival/death signals of Treg after different secondary polyclonal stimulations. Treg were stained with CFSE and stimulated with optimal strength of anti-CD3+ anti-CD28 microbeads or platebound anti-CD3+ soluble anti-CD28. CFSE dilution pattern, 7AAD staining and Bcl-2 expression of Treg population were assessed at day 4. Data are representative of four independent experiments.