Fig. 1: Defining the CD45⁺ immune microenvironment in pediatric T-ALL.

a Schematic overview of the single-cell CITE-seq (transcriptome and surface protein expression) and immune repertoire sequencing (TCRαβ/BCR-seq) workflow. The clinical cohort included fifteen pediatric T-ALL patients for whom samples were collected at diagnosis (Dx, PB (n = 10) and BM (n = 5)), short-term treatment (ST, PB (n = 8)) and remission (RM, PB (n = 6). Four healthy donor samples (PB (n = 2) and BM (n = 2)) were taken along as control samples. Samples were sorted for CD45^high immune cells and CD7⁺CD45^dim malignant cells. The 5’ 10x Genomics Immune Profiling sequencing protocol was used to analyze the transcriptome (RNA-seq), surface protein expression (ADT-seq, 204 surface proteins) and immune repertoire (TCRαβ/BCR-seq). b Weighted-nearest-neighbor (WNN) UMAP plot based on transcriptome and surface protein expression (CITE-seq) of 136,671 cells from fifteen T-ALL patients and four healthy donors, colored by cell type. mDCs: myeloid dendritic cells; pDCs: plasmacytoid DCs; NK: natural killer; HSPC: hematopoietic stem and progenitor cells. c Bubble heatmap showing normalized gene expression (left) and surface protein expression (right) of selected signature genes or proteins for cell types shown in (b). d Surface protein expression of CD34, CD1a, CD3, CD4 and CD8 in malignant cells and normal T cells from T-ALL patients at diagnosis. e Heatmap of average RNA expression of known T-ALL oncogenes¹⁵ in malignant cells of T-ALL patients at diagnosis. f Heatmap of percentage of cells with TCRβ motifs detected in the 10x Genomics scTCRαβ-seq data. g Distribution of immune cell types in healthy donors (BM: n = 2, PB: n = 2) and T-ALL patient samples (diagnosis: BM: n = 5, PB: n = 10; prophase: PB: n = 6; treatment: PB: n = 2, remission: PB: n = 6).