Both short and long demonstrate activity in vitro but only the short CAR is active in vivo. (A) Production of IFNγ and TNFα by short and long anti-TSLPR CAR T cells as measured by enzyme-linked immunosorbent assay of supernatant after coincubation with TSLPRhi ALL (REH TSLPR or MUTZ5). (B) Lysis of REH-TSLPR and MUTZ5 TSLPRhi ALL by both short and long CAR T cells measured by ⁵¹Cr release after 4-hour coculture. Results show specific lysis calculated as described in Methods. (C) Short TSLPR CAR T cells (15 × 10⁶) administered IV on day 4 after injection of luciferase-expressing REH-TSLPR demonstrate potent activity as measured by bioluminescent imaging, whereas long TSLPR CAR given at the same dose fail to alter leukemia progression. Control mice received the same dose of expanded GFP-transduced T cells. A single animal in the short TSLPR CAR group was sacrificed as the result of a wasting syndrome consistent with xenogeneic graft-versus-host disease. ACT, adoptive cell transfer. (D) In a separate experiment, mice were treated as in Figure 2C, and peripheral blood was analyzed on days 16 and 27 after injection of 15 × 10⁶ short or long TSLPR CAR T cells or GFP-transduced control T cells. Representative dot plots showing increased persistence of short CD8⁺ TSLPR CAR T cells compared with long CAR T cells on days 16 and 27 after injection. (E) Significantly increased absolute number of short TSLPR CAR T cells at day 27 after injection compared with long CAR T cells as measured using a bead calibration as described in “Methods.”