Earlier infusion of Tregs does not abrogate Tcon-mediated GvT effect. Lethally-irradiated Balb/c hosts received TCD-BMs and luc/yfp A20 leukemia cells, with or without, 1 × 105 Tregs from wt FVB, on day 0 followed by infusion of 1 × 106 Tcons 2 days later. Animals that received TCD-BMs and A20 cells alone served as controls. (A) Tumor engraftment is noted in bone marrow compartments (F indicates femur; H, humerus; S, sternum) and spleen (Sp) in all groups. Tumor progression is observed in animals that received TCD-BMs alone, while animals that received Tcons with or without Tregs cleared tumor by day 15. (B) Tumor clearance was stable, with no evidence of relapse with prolonged follow up by BLI at day 100 (□ versus • or □ versus ○, NS). Color bars in panel A represent signal intensity scale over whole body: (B) 3000 to 30 000 for all images of Tcons + A20 and Tcons + Tregs + A20, and day-5 image of TCD-BMs + A20; and 3 × 104 to 3 × 105, 3 × 105 to 3 × 106, and 3 × 106 to 3 × 107 for days 10, 15, and 20 images of TCD-BMs + A20 and (C) FACS analysis for yfp-expressing A20 cells in the bone marrow and splenic compartment 120 days following transplantation (dark gray fill is A20 cell positive control; light gray fill is BM negative control; black line is Tregs + Tcons + A20; red line is Tcons + A20). (D) Survival corresponded with BLI findings. Animals that received only TCD-BMs died from progressive tumor growth (n = 8); animals that also received Tcons (n = 9) cleared the tumor but died from progressive GvHD (▴ versus •, P < .001). Transplant recipients that received earlier infusion of low-dose Tregs (n = 9) were protected from leukemia (▴ versus ○, P < .001) and had significantly improved survival from GvHD (○ versus •, P = .004). Data were combined from 3 independent experiments.
