Fig 2: Activation of mTORC1 signaling decreases the CXCR4 level in EpCAM CAR-T cells. A, Fold-changes in transcription level of chemokine receptor genes in EpCAM CAR-T cell compared with control T cells, n = 3 human donors. B, FACS analysis of the proportion of CXCR4+ cells in control T cells and CAR-T cells. n = 4 normal human donors. Paired two-tailed Student t test. C, FACS analysis of CXCR4 expression in control T cells, T cells cultured with IL2 (100 U/mL), T cells stimulated with CD3/CD28 antibody (data shown as mean ± SD; one-way ANOVA with Tukey multiple comparisons test). D, FACS analysis of CXCR4 expression in T cells modified with control empty vector and EpCAM CAR-T cells. (data shown as mean ± SD; unpaired two-tailed Student t test). E, Transwell analysis of the counts of the control T cells (unmodified T cells) and EpCAM CAR-T cells that migrated to the lower chamber in response to CXCL12 (100 ng/mL). “-CXCL12,” without CXCL12 in the lower chamber; “+CXCL12,” with CXCL12 in the lower chamber (data shown as mean ± SD; unpaired two-tailed Student t test). Data shown are representative of three independent experiments. F, GSEA suggesting that EpCAM CAR-T cells are significantly enriched for the expression of transcripts with functional annotations related to the mTOR signaling pathway compared with control T cells (Enrichment plot: mTOR signaling pathway). G and H, FACS analysis of the phosphorylation of mTOR (G) and ribosomal protein S6 (H) in EpCAM CAR-T cells and control T cells, n = 3 normal human donors, paired two-tailed Student t test. I, Immunoblot analysis of mTOR signaling components in control T cells and EpCAM CAR-T cells, n = 3 normal human donors. Data are representative of two experiments. J, Transcription level of CXCR4 in EpCAM CAR-T cells treated with DMSO, rapamycin, everolimus, temsirolimus, JR-AB2–011, and KU0063794 for 24 hours, n = 3(data shown as mean ± SD ordinary one-way ANOVA with Tukey multiple comparison test). K, FACS analysis of the proportion of CXCR4+ EpCAM CAR-T cells treated with DMSO, rapamycin, everolimus, temsirolimus, JR-AB2–011, and KU0063794 for 24 hours, n = 3 (data shown as mean ± SD; ordinary one-way ANOVA with Tukey multiple comparison test). L, FACS analysis of the proportion of CXCR4+ EpCAM CAR-T cells treated with 0 nmol/L, 5 nmol/L, 10 nmol/L, 20 nmol/L, and 40 nmol/L rapamycin for 6 days. (n = 3, data shown as mean + SD; unpaired two-tailed Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns, not significant).