Single-cell sequencing reveals transcriptional and immunogenetic diversity in stereotyped chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) comprises immunogenetically defined stereotyped subsets of patients with distinct B-cell receptor (BcR) immunoglobulin features and clinical trajectories, yet the molecular pathways underlying subset-specific differences remain incompletely characterized. To resolve disease-relevant heterogeneity obscured in bulk analyses, we performed integrated single-cell transcriptomic and immunogenetic profiling of 48 557 malignant and bystander immune cells from 13 treatment-naïve primary patient samples representing poor-prognostic subsets #1 and #2 and the indolent subset #4. Despite interpatient variability, leukemic cells exhibited pronounced subset-specific transcriptional features, with enrichment of hypoxia-related genes in subset #1, oxidative phosphorylation (OXPHOS), MYC/E2F targets, and mechanistic target of rapamycin complex 1 (mTORC1) signaling in subset #2, and negative enrichment of hypoxia, apoptosis, and reactive oxygen species-related pathways in subset #4. Notably, compared with indolent subset #4, aggressive subsets #1 and #2 harbored increased proportions of metabolically active and recently emigrated/proliferative leukemic cells, characterized by a CXCR4dim CD5bright transcriptional phenotype and concerted enrichment of MYC target genes, mTORC1 signaling, OXPHOS, and BcR signaling. Immunogenetic analyses revealed dominant malignant clones with evidence of intraclonal immunogenetic diversification in 11 of 13 cases. T cells were dominated by effector-memory phenotypes exhibiting progressive differentiation toward terminal cytotoxic states, accompanied by exhaustion-associated programs, and expanded T-cell clones largely restricted to terminal states. Ligand-receptor analysis further indicated costimulatory and inhibitory signaling between leukemic and immune cells. Together, this study provides an integrated view of the transcriptional and immunogenetic landscape of 3 major stereotyped CLL subsets, linking BcR immunoglobulin configuration with proliferative capacity and microenvironmental cross talk, thereby shaping clonal behavior.