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case_images: Study overview. Combined scRNA sequencing of peripheral blood and spatial omic profiling of tissue across time provide a global view of progression in a patient with cutaneous T-cell lymphoma. (A) For scRNA sequencing, combined 10x genomics-based 5'and TCRalpha/beta sequencing workflows were used. (B) For spatial omic analysis, the digital spatial profiling GeoMx platform was applied.
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case_images: Transcriptionally distinct T-cell subsets. (A) CD3+ T-cells were separated into 16 distinct clusters through scRNA sequencing and are colored by the cluster number to the left, and by time point to the right. (B) One key differentially regulated gene per cluster, in total 16, is visualized in each feature plot and reveals biological differences between the clusters. The scale represents log-normalized expression values. Expression values below 1 and above 2 are truncated.
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case_images: Cell classification of individual clusters. (A) A semi-automated approach was used to annotate the clusters, and nine phenotypically defined cell types were defined. The clusters are colored by the cluster and cell type label. (B) The number of total cells in the three time points in each cluster across the identified cell types. (C) The relative proportion of cells in the three time points in each cluster (0-15).
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case_images: TCR sequencing reveals CD4+ and CD8+ expanded clones. The colors represent the clonotype genes indicated at the lower right panel. (A) The relative proportion of the top seven clonotypes among total number of cells at baseline displayed in one color each. Clonotypes only detected at a low proportion are jointly displayed in grey, and unmapped cells are displayed in white. (B) Clonotype identity of each cell in the integrated UMAP plot. The small black dots represent the unmapped cells with no T-cell receptor alpha/beta chain. (C) The relative proportion of each of the top seven clonotypes across the clusters at baseline. (D) The relative proportion of each of the top seven clonotypes across the different cell types at baseline. (E) The percentage of TCR sequenced cells of each of the top seven clonotypes, visualized across the three time points (baseline, progression 1, and progression 2). (F) The relative proportion of the top seven clonotypes in each cluster, visualized across the three time points (baseline, progression 1, and progression 2).
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case_images: Differentially regulated genes (DEGs) at baseline. DEGs associated with different expanded compared to non-expanded populations were determined. (A) Venn diagram of unique and common genes across C5_CD4_TE, C10_CD4_TE, expanded CD4_EM, and expanded CD8_TE. (B) Volcano plot of the 13 unique DEGs associated with the progression-related C5_CD4_TE. (C) Volcano plot of the 50 unique DEGs associated with malignant population C10_CD4_TE. (D). Dot plot of the 21 common genes among C5_CD4_TE and C10_CD4_TE but that differ compared to non-expanded CD4 cells. Red boxes indicate key genes. (E) Volcano plot of the 16 unique DEGs associated with expanded CD4_EM cells. (F) Volcano plot of the 48 unique DEGs associated with expanded CD8_TE cells. Genes marked with red and blue boxes indicate important features for each expanded T-cell population.
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case_images: Pathway analysis comparing expanded peripheral T-cell populations compared to non-expanded cells. The top five identified pathways in each comparison driven by differentially regulated genes; the top significant (adjusted p-value < 0.05) pathways are ranked based on absolute normalized enrichment score (abs(NES)). The following comparisons were made: C5_C10, Exp_CD4 (C9 and C11), C10, and C5 were each compared to non-expanded CD4 cells (C6, C7, C12, C15). Exp_CD8 (C4 and C8) were compared to non-expanded CD8 cells (C2 and C3).
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case_images: Differentially regulated genes (DEGs), associated with progression of MF. Key genes associated with C5_CD4_TE are marked with red boxes. (A) Dot plot of top 10 DEGs associated with progression in C5_ CD4_TE. (B) Dot plot of top 10 DEGs associated with progression in C10_ CD4_TE. (C) Top 10 unique genes associated with progression in C5_ CD4_TE. Targeted analysis of CTCL-related markers in (D) C5_ CD4_TE and (E) C10_CD4_TE. Targeted analysis of Th1- and Th2-related immune markers in (F) C5_ CD4_TE and (G) C10_CD4_TE.
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case_images: Tissue-based analysis reveals intra-tumor heterogeneity at early progression. (A) Different regions were defined as either close to the epidermis (pink box) or far from the epidermis (orange box) in the tissue biopsy at progression 1. (B) Common DEGs in CD3+/CD8- and CD3+/CD8+ cell segments when the two spatial localizations are compared. (C) Unique DEGs for each of the CD3+/CD8- and CD3+/CD8+ cell segments, respectively, when the same two spatial localizations are compared.
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case_images: Differentially regulated genes differentiating 1st and 2nd progression. (A) Venn diagram showing the number of unique and common DEGs comparing progression 1 and 2 in the three cell type segments. DEGs with higher expression at progression 1 are shown to the left, and DEGs with higher expression at progression 2 are shown to the right. (B) Volcano plot of all DEGs identified for each of the cell segments. DEGs with higher expression at progression 1 are shown in red to the right, and DEGs with higher expression at progression 2 are shown in blue to the left for (B) CD3+/CD8-cell segments, (C) CD3+/CD8+ cell segments, and (D) CD163+ cell segments. (E) Top five identified pathways driven by the identified DEGs in targeted cells in the immune microenvironment in MF tissue cells comparing progression 1 and progression 2. Volcano plots of DEGs that are unique for each of the cell segments are shown in (F) for CD3+/CD8- cell segments, in (G) for CD3+/CD8+ cell segments, and in (H) for CD163+ cell segments. Genes marked in red and blue indicate features associated with the CD3+/CD8- cells.
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case_images: Targeted analysis. (A) Targeted analysis of CTCL markers (same panel as in Figure 7D,E), shown as box plots comparing progression 1 (red) and progression 2 (green) for each marker separately. (B) Selected genes associated with targeted CCR4 and CD30 treatment. Expression of CCR4 and CD30/TNFRSF8 across the two time points in (B) CD3+/CD8- cell segments. Expression of CCR4 and TNFRSF8/CD30 across the three cell segments at (C) progression 1 and (D) progression 2. (E) Targeted analysis of markers associated with a shift from Th1 to T2 immune profile. Comparison of expression of individual markers (same panel as in Figure 7F,G) in progression 1 (red) and progression 2 (green) in the CD3+/CD8- cell segments.
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