CAR T cells given on day 0 compared with day +5 have higher early expansion associated with a more activated phenotype. (A) To study fresh CAR T cells but also remove any confounder of differences between different CAR T-cell products, 2 separate transplantations were aligned such that on the same day mice in 1 transplantation received CAR T cells on day 0 and mice in the other transplantation received on day +5 cells from the same CAR T-cell product. Splenocytes for transduction came from CD45.1⁺CD45.2⁺ B6C3F1 mice to most definitively separate CAR⁺ (Protein-L⁺CD45.1⁺) from graft-derived (CD45.1⁻) T cells as wild-type (CD45.1⁻CD45.2⁺) B6C3F1 mice were used for the splenocytes and bone marrow for transplant. Recipient mice were euthanized and assessed by flow cytometry 3, 8, or 16 days post infusion of CAR T cells. (B-D) Shown are (B) total numbers and (C) percentages of viable donor CAR T cells at each time point as well as (D) the relative distribution of CD4⁺ vs CD8⁺ CAR T cells. CAR T cells administered on day 0 had better early expansion 3 days post infusion, whereas CAR T cells administered on day +5 had better expansion at 8 and 16 days post infusion, particularly within the CD4⁺ CAR T cells. (E) More robust recovery of CD4⁺ CAR T cells with a regulatory T-cell phenotype (CD25⁺Foxp3⁺) was seen in mice receiving CAR T cells on day +5. (F) CAR T cells given on day 0 had similar to higher proliferation at all time points. (G-H) Most consistently seen within the spleen, there was significantly higher PD1 and LAG3 expression at early time points for CAR T cells given on day 0 compared with on day +5. (I) Greater differentiation (higher CD62L⁻) was seen at 8 and 16 days after infusion for CAR T cells given on day 0 vs CAR T cells given on day +5. Combined results of 2 independent experiments are shown with n = 5 mice per group per experiment. Data underwent natural logarithmic (total numbers) or arcsin (percentages) transformation prior to t test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.