Supplementary Figure 3: Ad5f35 transduction leads to a pro-inflammatory (M1) macrophage phenotype.

a, Hierarchical clustering of differentially expressed genes from UTD or Ad5f35-CAR-HER2-ζ transduced human macrophages from (n) = 4 matched donors, 48 hours post transduction. The heatmap shows log₂ fold-change in gene expression relative to UTD. b, Table of Ad5f35 induced canonical pathways in human macrophages, derived from (n) = 4 matched donors. Statistical analysis was performed using Fisher’s exact test. c, Heatmap of differentially expressed co-stimulatory ligands, antigen processing genes, and MHC-I/MHC-II genes between UTD and Ad5f35 transduced CAR macrophages via RNA-seq. d, Confirmation of select RNA-seq results in (3c) via RT-qPCR. Data represent the mean ± SEM of (n)=3 technical replicates. Statistical analysis was performed using one-way ANOVA with multiple comparisons. e, Mean fluorescence intensity of human M1 markers CD80 and CD86 and M2 marker CD163 in response to transduction with increasing MOIs of Ad5f35-CAR by FACS. Data is represented as mean ± SEM of (n)=2 technical replicates. Experiment was repeated at least 3 times. f, Surface expression of human M1 markers (CD80 and CD86) and M2 marker CD163 after transduction with equivalent MOIs of control empty-vector Ad5f35 or Ad5f35-CAR. Data is represented as mean ± SEM of (n)=2 technical replicates. Experiment was repeated at least 3 times. g, Surface expression of M1 marker CD86 on control UTD or Ad5f35-CAR transduced macrophages from (n)=10 human matched-donors. h, Persistence of M1 marker expression (CD80 and CD86) on primary human UTD or CAR macrophages from (n)=3 human donors over the course of 40 days of in vitro culture. For all panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.