Treg cell lines generated from cord blood exhibit impaired proliferation and IL-2 production in response to stimulation by TCR and costimulation. (A) Treg and Tcont cell lines were stimulated with anti-CD3/CD28–coated dynal beads (3 + 28) for indicated time intervals. Proliferation was assessed by [3H] thymidine incorporation. (B) Secretion of IL-2 was analyzed by ELISA. (C) Supplementation with IL-2 (50 U/mL) induces proliferation of Treg cell lines stimulated with anti-CD3/CD28 beads. Treg and Tcont cell lines were cultured as in panel A, but IL-2 (50 U/mL) was added during culture. (D) Tregs are markedly hyporesponsive to stimulation with allogeneic DCs compared with Tcont cell lines. Tcont and Treg cell lines were stimulated with allogeneic DCs as described in “Materials and methods” for the indicated time intervals. Proliferation was assessed by [3H] thymidine incorporation. (E) Treg hyporesponsiveness mitigated by IL-2 supplementation. Treg and Tcont cell lines were cultured as in panel D, but IL-2 (50 U/mL) was added during culture. (F) Treg cell lines do not respond to IL-2 alone as do Tcont cell lines. Treg and Tcont cell lines were cultured in various concentrations of IL-2 (5-500 U/mL) and proliferation was assessed by thymidine incorporation, with peak responses shown after 2 days of culture. Results are all representative of 3 to 4 independent culture experiments. cpm indicates counts per minute.