GVT effector function of T_CONVcells is preserved in the presence of Treg cells and RAPA. Tumor growth and elimination are visualized by bioluminescence imaging (A), FACS analysis (B), histology (C), emitted photons over time (D), and survival (E). All Balb/c recipients (H-2k^d) were given 1 × 10⁵ A20 leukemia cells (H-2k^d) and BMT was performed as described in “Materials and methods.” (A) luc⁺A20 tumor-cell localization at 5 days (top row), 10 days (middle row), and 15 days (bottom row) after BMT in representative animals given transplants with TCD-BM alone (first column from left), TCD-BM plus T_CONV cells (second column from left), TCD-BM plus T_CONV and Treg cells (third column from left), and TCD-BM plus T_CONV and Treg cells and RAPA (fourth column from left). ST indicates sternum; HU, humerus; FE, femur; SPL, spleen. (B) Bone marrow samples with yfp⁺ A20 cells of recipients on day 12 after transplantation with TCD-BM alone (first column from left), TCD-BM plus T_CONV cells (second column from left), TCD-BM plus T_CONV and Treg cells (third column from left), and TCD-BM plus T_CONV and Treg cells and rapamycin (fourth column from left). (C) H&E stains of representative spleen samples gathered on day 12 from animals receiving TCD-BM only (Ci), TCD-BM plus T_CONV cells (Cii), TCD-BM plus T_CONV and Treg cells (Ciii), and TCD-BM plus T_CONV and Treg cells and RAPA (Civ). A homogenous leukemic infiltration is found only in animals receiving TCD-BM only (Ci) but not in the other groups. Microscopic evaluation was performed on a Nikon Eclipse TE 300 microscope equipped with 20 ×/0.45 and 40 ×/0.60 objective lenses (Nikon, Melville, NY). Photographs were captured by using a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI). Digital images were saved as TIFF files, which were inserted into and processed with PowerPoint software (Microsoft, Redmond, WA). (D) Expansion of luc⁺A20 tumor cell as measured in photons over total body area (photons/second/mouse). Animals rejecting the A20 tumor demonstrate a rapid signal loss within the first 15 days after BMT. (E) Survival of BALB/c mice receiving TCD-BM alone (▵, n = 15), TCD-BM plus A20 leukemia cells (□, n = 15), TCD-BM plus A20 leukemia cells plus T_CONV cells (•,n = 15), TCD-BM plus A20 leukemia cells plus T_CONV and Treg cells (⋄,n = 12), or TCD-BM plus A20 leukemia cells plus T_CONV and Treg cells and RAPA 0.5 mg/kg; ○,n = 12). Data from 3 independent experiments are combined.