Single-cell RNA sequencing analysis of patient leukemia. (A) Schematic of single-cell RNA-sequencing experimental design and data processing pipeline. (B) Uniform manifold approximation and projection (UMAP) of combined patient data annotated by patient. (C) UMAP of combined patient data annotated by genotype, determined by the souporcell algorithm. (D) UMAP of combined patient data annotated by patient malignant cells and cell type, determined by (B-C) and viewmastR algorithm, respectively. (E) Network map of significantly enriched Gene Ontology (GO) Biological Processes 2021 pathways (P < .05) in genes upregulated (log2FC ≥ 0.25) in blasts after HA-1 TCR-T compared with before the treatment in all 3 patients with preinfusion and postinfusion samples. Bubble size corresponds to larger –log (average adjusted P value) for each pathway. Blue, neutrophil-related pathways; red, cell death/survival; yellow, cytokine signaling; green, metabolism; gray, other processes. (F) Bubble plots illustrate the expression patterns of 5 genes. These genes were identified from the significantly enriched pathways and had a greater log2 fold-change after the infusion in malignant cells compared with the change in nonmalignant cells (log2FC malignant ≥1.5× log2FC normal) in all 3 patients with pre- and post-HA-1 TCR-T samples. (E-F) Postinfusion sample time points: patient 4, day 29; patient 6, day 18; patient 9, day 90. viewmastR images in panel A are from https://github.com/furlan-lab/viewmastR/blob/main/man/figures/viewmaster.png.