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. 2020 Jun 10;8(1):e000845. doi: 10.1136/jitc-2020-000845

Figure 3.

Figure 3

Both 41BB-CD123 and CD28-CD123 CARTs eliminate healthy CD34+ HSPCs in vitro and in vivo. (A) Experimental scheme for in vitro assessment of CAR123 cytotoxicity on healthy CD34+ cells. (B) Representative FACS showing the residual CD34+ HSPCs (red) after exposure to CAR123 CARTs or MOCK T- cells for 72 hours at an E:T ratio of 2:1. (C) Absolute quantification of remaining alive CD34+ cells after exposure to either 41BB-CD123 or CD28-CD123 CARTS (72 hours, E:T 2:1). (D) Clonogenic assays performed with residual alive CD34+ HSPCs after 24 hours of coincubation with either 41BB-CD123 CARTs, CD28-CD123 CARTs or MOCK T-cells (E:T 1:1) (n=3 donors). (E) Schematic representation of the in vivo experimental plan. CD34+ cells were intra-BM transplanted into NSG mice, and 6 weeks later, the level of human engraftment was assessed by FACS analysis in PB and BM. Mice then received 3×106 of either CD123 CARTs (41BB or CD28) or MOCK T-cells. PB bleedings were performed biweekly and PB/BM were analyzed at sacrifice (6 weeks after CART infusion). (F, G) Analysis of murine PB (F) and BM (G) multilineage reconstitution (CD19+ B lymphoid, CD123+ myeloid and CD34+ immature) at the indicated weeks post-CART infusion. Final engraftment (POST) of myeloid, B lymphoid and immature HSPCs is presented as fold change in comparison to pre-CART/MOCK infusion (PRE). (H) Schematic representation of the in vivo experimental plan. CD34+ cells were intra-BM transplanted into NSG mice, followed the day after by infusion of either 3×106 CD123 CARTs (41BB or CD28) or MOCK T-cells. Mice were sacrificed 6 weeks after and PB and BM were analyzed. (I, J) Analysis of murine PB (I) and BM (J) multilineage reconstitution (CD19+ B lymphoid, CD123+ myeloid and CD34+ immature) 6 weeks after CART infusion. *P<0.05, **P<0.01, ***P<0.001. BM, bone marrow; CART, chimeric antigen receptor T-cell; E, erythroid colony-forming unit; E:T, effector:target; FACS, fluorescence-activated cell sorting; G, granulocytic colony-forming unit; GEMM, granulocytic, erythroid, myelomonocytic colony-forming unit; GM, granulomonocytic colony-forming unit; HSPC, hematopoietic stem/progenitor cell; M, monocytic colony-forming unit; NSG, non-obese diabetic-Cg-Prkdcscid Il2rgtm1Wjl/SzJ; PB, peripheral blood.