Fig. 1.

Murine CD62L⁺ Treg retain their phenotype and function during in vitro expansion and express gut homing receptors. CD4⁺CD25^highCD62L⁺ Treg and CD4⁺CD25⁻ Tconv were FACS-sorted for in vitro expansion. a Gating strategy for sorting of CD4⁺CD25^highCD62L⁺ Treg from BALB/c splenocytes and a representative example for sort purity according to CD4, CD25, and CD62L expression. b Expansion rates of FACS-purified BALB/c and C57BL/6 Treg (n = 20 independent cultures each). c Foxp3 expression of sorted Tconv and CD62L⁺ Treg after 14 days of in vitro culture. Representative FACS plots and summarized data of ten independent cultures. d Expression of Helios, Neuropilin, CD62L, CCR7, and the gut homing receptors CD103 and LPAM-1 on CD62L⁺ Treg before and after 14 days of in vitro culture (n = 10 independent cultures, *p < 0.05, **p < 0.01, ***p < 0.001). e Suppressive activity of Treg before and after in vitro expansion. Freshly isolated CFSE-labeled CD4⁺CD25⁻ Tresp cells were stimulated with anti-CD3ε in the presence of irradiated, autologous APC, and freshly sorted (combined data from five independent experiments) or in vitro expanded Treg (combined data from eight independent experiments) at the indicated ratios for 3 days. In vitro expanded Tconv served as negative control. Proliferation of Tresp cells was determined by FACS and is shown as % of proliferation observed in cultures with Tresp alone. All summarized data are shown as mean ± s.e.m