Fig. 3: DCs are required for enhanced persistence of CAR T cells in irradiated tumors.

a, Zbtb46^DTR/DTR nongenotoxic BM chimeras inoculated with 7 × 10⁵ target⁺ orthotopic KP lung tumors through TVI were randomized 3 weeks later to 8 Gy of TRT or no irradiation. Cohorts were then lymphodepleted and treated with Luciferase and CAR double-transduced syngeneic mouse T cells to a dose of 2.5 × 10⁶ CAR T cells per mouse. To ensure sustained depletion of DCs, DT was administered starting the day before CAR T cell injection and every 2–3 days after for the remainder of the experiment in the cohorts indicated. Kinetics of thoracic CAR T cell persistence by BLI were determined. Faint lines track bioluminescence from individual mice. Median values are shown in bold (n = 6 for no DT + 8 Gy, n = 7 for DT + 8 Gy and n = 5 for 0 Gy). Day 17, P = 0.0140; day 23, P = 0.0426; day 28, P = 0.0182, Mann–Whitney U-test. b,d, Representative flow cytometry plots (b) and quantification (d) of DsRed⁺ CAR T cells in the lungs of target⁺ KP tumor-bearing mice isolated 9 days after adoptive cell transfer and stained for expression of indicated surface markers. Zbtb46^DTR/DTR or WT BM chimeras were inoculated with 7 × 10⁵ target⁺ orthotopic KP lung tumors through TVI then treated 3 weeks later with 8 Gy of TRT. Cohorts were then lymphodepleted and treated with syngeneic T cells transduced with a CAR-2A–DsRed retroviral construct to a dose of 2.5 × 10⁶ CAR T cells per mouse. To ensure sustained depletion of DCs, DT was administered starting the day before CAR T cell injection and every 2–3 days after to both groups of mice (n = 5 for WT + 8 Gy, n = 6 for Zbtb46^DTR/DTR + 8 Gy). Statistical analysis conducted using a two-sided t-test. c, Representative paraffin sections of target⁺ KP tumor-bearing lungs isolated 9 days after CAR T cell infusion, stained for DsRed expression on CAR T cells (example indicated by black arrowhead; hematoxylin counterstained). Scale bars, 100 µm. e, Quantification of intratumoral DsRed⁺ CAR T cell density 9 days after infusion of CAR T cells. Each dot represents a tumor nodule from representative lung sections from representative lung sections from n = 5 (WT + 8 Gy, 33 tumors) or n = 6 (Zbtb46^DTR/DTR + 8 Gy, 40 tumors) mice per irradiated group (n = 4–10 tumors per lung section). Bars illustrate mean values. ***P = 0.0007, ordinary one-way ANOVA.

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