Fig. 4. Single-cell transcriptome analysis combined with deep sequencing for characterization of subclonal population.

a Workflow shows the collection and processing of fresh peripheral blood samples from indolent ATL, followed by scRNA-seq/deep sequencing platform. b Line plot shows transition of VAFs at T1 to T2 in CD4⁺/CADM1⁺/CD7⁻ population in ATL#6. Each line represents transition of VAF values of mutated genes during T1 to T2 period. Putative driver genes in Clone A (blue) and Clone B (orange) are annotated. c t-distributed stochastic neighbor embedding (t-SNE) projection from P, D, and N subpopulations (total 13,087 cells) reveals graph-based clusters. The clusters are assigned to the indicated phenotypes by cell barcode ID. d t-SNE plots with single-cell mutation detection. Cells colored according to single-cell genotype at Clone A genes (VAV1^Y174C, IKBKB, and GPR183) and Clone B genes (VAV1^M501R/V and TBL1XR1): blue and red, at least one mutant read detected; yellow, wild-type reads only; gray, no coverage. e t-SNE plots with viral antisense RNA (left) and clone-specific chimeric transcripts at Chr16 and Chr2 (right). f t-SNE plot shows large clusters (C1 to C5) as identified by differentially expressed marker genes, distribution of cell phenotype, detection of mutated RNA and clone-specific chimeric RNA, and population size estimated by VIS reads. g Clustered heatmap depicts expression levels (Log₂ fold change) of TCR pathway target genes. h Box plot shows normalized average of expression levels of TCR pathway target genes (167 genes from GSE13738 dataset) in clustered populations. The middle lines within box plots correspond to the medians; lower and upper hinges correspond to first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR. All data points are overlaid on the box plot. *p ≤ 0.05 (two-sided Student’s t test). i Bar graph shows reporter-based NFAT activity in Jurkat cells expressing wild-type or mutant VAV1 in the presence of TCR engagement. n = 3–4 biologically independent samples, mean ± SD, *p ≤ 0.05 (two-sided Student’s t test). Raw data are available from Source data file. j Bar graph shows enriched KEGG pathways with one-sided Fisher’s exact p values (−Log₁₀) in C3 cluster cells compared with C2 (Log₂FC ≥ 2 in C3, 2234 genes). k Venn diagram depicts overlapped and clone-specific genes in Clone A and Clone B at T1 and T2 (FC ≥ 2 vs. P subpopulation, p ≤ 0.05). l, m Box plots show normalized Log₂ fold changes of TCR pathway target genes (167 genes from GSE13738 dataset) (l) and cell-cycle process genes (377 genes) (m) in clustered subpopulations at T1 and T2. P, uninfected T cells. The middle lines within box plots correspond to the medians; lower and upper hinges correspond to first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR. All data points are overlaid on the box plot. *p ≤ 1 × 10⁻⁵ (two-sided Student’s t test).