Fig. 6: Single-cell RNA sequencing reveals a CXCR4-driven memory signature in CAR-T cells from AML PDX.

a Schematic of experimental design detailing CAR-T cell collection for single-cell RNA sequencing. Cells were obtained from AML PDX BM, spleen, and liver at 2.5 weeks, 4 weeks, and 5 months post-injection. b UMAP projection of BM-derived CD25-targeted CAR-T cells collected at 4 weeks and 5 months post-injection. Cells are colored by CD4/CD8 identity, cell cycle score, and select memory/exhaustion markers. Cell type annotations were made based on canonical T-cell subtype markers (see Fig. S7c). c Relative abundance of each cluster across treatment conditions. Forest plot shows log₂ fold-enrichment of each subtype in CXCR4(-) or mCXCR4(+)treatment groups (n = 3 animals per group) at 4 weeks, with 95% confidence intervals. Diamonds indicate mean; whiskers represent 95% confidence intervals. Statistical comparisons were made using two-tailed t-tests. Non-adjusted p-values are reported. See also Figure. S7e. d Dot plot showing expression of representative marker genes across T-cell subtypes and treatment conditions. Additional markers and full subtype profiles are shown in Figure. S7f. e Differential expression (DE) analysis comparing CXCR4(-) versus mCXCR4(+) CD25-targeted CAR-T cells at 4 weeks and 5 months. Genes with base mean >15, FDR < 0.05, and log₂ fold-change ≥ 0.58 in at least one comparison are shown. Genes with early (4-week) or late (5-month) CXCR4-associated upregulation are highlighted in red and blue, respectively. f Gene set enrichment analysis (GSEA) of DE genes ranked by log₂ fold-change. GSEA p-values were calculated using the fgsea package in R. FDR-adjusted p-values are reported. Only pathways with FDR < 0.05 in at least one contrast and enrichment FDR < 0.01 are displayed. g UMAP visualization of liver- and spleen-derived CAR-T cells harvested at 2.5 weeks post-injection. Cells are colored by CXCR4 expression status and tissue origin. ALRA-imputed expression of select memory and exhaustion markers is shown. Source data are provided as a Source data file for (c, f). “Fig. 6a” created in BioRender. Liang, M. (https://BioRender.com/j4jhcy1) licensed under CC BY 4.0.