Figure 3.

CART33 and CART123 cells exhibit antitumor activity in MDS. (a) CART33 and CART123 cells undergo specific degranulation in response to bone marrow cells from MDS patients. Bone marrow samples from MDS patients were CD34 enriched (≥85% purity) and then incubated with CART33, CART123 or UTD cells at E:T ratio of 1:5 for 4 h, in the presence of CD49d, CD28 co-stimulation and monensin. CD107a degranulation was then measured by flow cytometry. (b) CART33 and CART123 cells kill CD34-enriched bone marrow cells from MDS patients. CD34-enriched bone marrow from patients with MDS were incubated with either UTD, CART33 or CART123 for 24 h, and then live leukemic cells were measured by flow cytometry. There was a significant reduction in live CD45^dimCD34+ cells in samples treated with CART33 or CART123. (c) Treatment with CART33 results in specific killing of the MDS clone. CD34-enriched bone marrow sample from a patient with MDS and 5q deletion was incubated with CART33, UTD cells or with no treatment at 1:1 E:T ratio for 4 h. Sample was then harvested, and fluorescence in situ hybridization for 5q- was performed. There was significant reduction in the 5q- clone percentage in the group treated with CART33 when compared with UTD and No treatment groups (as T cells comprised 50% of the sample, interphase nuclei were corrected by a factor of two). Results are representative of three experiments.