Fig 1: 4H1128ζ-IL12 CAR T cells phenotypically alter and delete tumor associated macrophages. (a) Peritoneal washes from tumor-bearing mice treated with 4H1128ζ-IL12 compared to 4H1128ζ CAR T cells and assessed for F4/80+ CD11b+ TAM (*p < 0.05). Data are plotted as mean ± SEM. Data shown are pooled results from 3 independent experiments. (b) Statistically significant (p < 0.05) differential gene expression between F4/80+ CD11b+ TAM recovered from mice treated with 4H1128ζ-IL12 vs 4H1128ζ. mRNA was extracted from pooled TAM from 2 mice per group after FACS sorting. n = 6 (4H1128ζ), n = 6 (4H1128ζ-IL12). Data presented as fold change of 4H1128ζ-IL12 TAM / 4H1128ζ TAM and plotted as mean ± SEM. (c) Analysis of Fas expression on F4/80+ TAM recovered from animals treated with CAR T cells (*p < 0.01). Data are plotted as mean ± SEM. Data shown are pooled results from 3 independent experiments. (d) TAM recovery from day 35 tumor-bearing mice treated with anti-FasL (αFasL) neutralizing antibody 1 day prior to CAR T infusion, *p < 0.01. Data are plotted as mean ± SEM. Data shown are pooled results from 3 independent experiments. (e) Female WT mice age 6–8 weeks inoculated with 1 × 107 ID8-Muc16ecto cells i.p followed by treatment with 250 μg αFasL i.p on day 41 and CAR T cells on day 42. *p = 0.04. Number of mice per group: 5 (PBS), 5 (αFasL), 6 (PBS + 4H1128ζ), 6 (αFasL + 4H1128ζ), 7 (PBS + 4H1128ζ-IL12), 6 (αFasL + 4H1128ζ-IL12). Results are pooled from 2 independent experiments. (f) WT tumor-bearing female mice between 6–8 weeks old were treated with 2 doses of clodronate (Clod) or PBS liposomes on Day 38 and 40. Subsequently, mice were treated with CAR T cells on D42. *p < 0.05. Number of mice per group: 5 (PBS), 5 (Clod), 5 (PBS + 4H1128ζ), 5 (PBS + 4H1128ζ-IL12), 6 (Clod + 4H1128ζ), 7 (Clod + 4H1128ζ-IL12), 5 (Clod + 1928ζ), 5 (Clod + 1928ζ-IL12). Results are pooled from 3 independent experiments. Statistical analysis performed using a log-rank (Mantel-Cox) test.