Supplementary Figure 5: Ad5f35 transduced CAR-M convert M2 macrophages toward M1 and activate T cells.

a, Principle component analysis plots demonstrating the phenotypic shift of M2A, M2C, or M2D macrophages after treatment with control UTD macrophage conditioned media (red) or CAR macrophage conditioned media (blue). Data was derived from (n)=3 technical replicates. b, Evaluation of surface expression of M1 markers CD80 and CD86 or intracellular expression of the immunosuppressive cytokine TGF-β on M2A, M2C, or M2D macrophages after treatment with UTD or CAR CM for 48 hours. Data represent mean ± SEM from (n)=3 technical replicates; statistical significance was calculated with a two-tailed t-test. c, Real-time PCR confirmation of expression of NY-ESO-1 after transduction of SKOV3 cells with a lentivirus co-expressing NYESO and HLA-A201. Data represents mean ± SEM of (n)=3 technical replicates. Experiment was performed twice. d, Representative FACS plot confirming HLA-A201 expression after transduction of SKOV3 cells with a lentivirus co-expressing NYESO and HLA-A201. Experiment was performed twice with similar results. e, Control or NY-ESO-1 expressing macrophages (No Ag and Ag, respectively), with or without Ad5f35-CAR were co-cultured with CTV-labeled anti-NY-ESO-1 T cells. Proliferation of anti-NY-ESO-1 TCR+ CD8+ T cells is shown as mean ± SEM of (n)=3 technical replicates; statistical significance was determined using ANOVA with multiple comparisons. f, Tumor burden of NSGS mice with metastatic SKOV3 that were treated IV with CAR-M alone, CAR-M + donor derived non-engineered/non-expanded T cells, or T cells alone, 63 days post treatment. Data represents mean ± SEM from (n)=3-5 mice per group, as indicated in the figure; experiment was performed once; statistical analysis was calculated with a Mann-Whitney U test. For all panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.