Characterization of transcriptional heterogeneity between patients with CTCL by paired scRNAseq and scTCRseq analyses of CD4⁺T cells. CD4⁺ T cells purified from the peripheral blood of 11 patients with CTCL underwent paired single-cell mRNA and TCR VDJ sequencing. (A) Summary of study design including sample preparation, sequencing, multidimensional data analysis, and mechanistic studies. (B) TCR clonotype frequency in purified CD4⁺ T cells from patients with CTCL (P1-P11) and healthy controls (N1-N3). Dominant clonotype: most frequent complete TCRα and TCRβ CDR3 transcripts. Dominant clonotype–like: TCRα CDR3 or TCRβ CDR3 matching the top clonotype in the absence of any transcript for the other chain. Other clonotypes: all other TCRα and/or TCRβ CDR3 transcripts. Note that in P6, 2 complete TCRα and 2 complete TCRβ sequences were present in the dominant clonotype. (C) A UMAP resulting from the integration of the scRNAseq transcriptomes of samples derived from 11 patients with CTCL (112 840 single cells distributed by annotated, unsupervised clustering) highlights interpatient diversity (each patient with CTCL is distinctly colored), as well as commonality of normal CD4⁺ T cells among the different patients (blue). (D) Total CD4⁺ T cells from each patient are individually highlighted throughout the integrated UMAP: normal CD4⁺ T cells (blue), CTCL cells (highlighted in red according to the dominant CDR3 sequence of each patient). (E) T-cell subsets based on characteristic gene expression are highlighted throughout the integrated UMAP. Th2-like: GATA3 >1, CCR4 >1; Treg-like: FOXP3 >1, CTLA4 >1; Tfh-like: PDCD1 >1, CXCR5 >1; Th17-like: RORC >1, CCR6 >1. (The gene cutoff of 1 indicates that the normalized and scaled gene expression is >1.) UMAP, Uniform Manifold Approximation and Projection.