Sézary syndrome originates from heavily mutated hematopoietic progenitors

Abstract

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

Graphical abstract

Figure 1.

Gene expression patterns in SS and MF. (A) Uniform manifold approximation and projection (UMAP) of combined analysis of single-cell RNA sequencing (scRNA-seq) from sorted CD4⁺CD26⁺ and CD4⁺CD26⁻ isolated T cells from peripheral blood of patients with SS SS1-SS4 (n = 4) and patients with MF MF1-MF3 (n = 3) by group (n = 7). (B) UMAP plots of each MF and SS major and minor clones and occupation of cells in each group with bar graph (%) representative of percent of cells in each group. (C) Trajectory analysis and pseudotime (slingshot) analysis of SS cells (top) compared to MF cells (bottom). (D) Bubble plot of stemness score and exhaustion score colored by Shannon clonality index and sized by cell count of 7 groups. Bubble plot of single-cell ATAC-seq open chromatin regions and scRNA-seq gene expression patterns across phenotypes (stem-like, transition, effector, progenitor exhaustion, proliferation, mitochondrial activity, and T-regulatory) in SS (E) and MF (F). Color of each dot represents normalized average gene expression from high (red) to low (blue). Size of each dot represents the percentage of positive cells for each gene.

Figure 1.

Gene expression patterns in SS and MF. (A) Uniform manifold approximation and projection (UMAP) of combined analysis of single-cell RNA sequencing (scRNA-seq) from sorted CD4⁺CD26⁺ and CD4⁺CD26⁻ isolated T cells from peripheral blood of patients with SS SS1-SS4 (n = 4) and patients with MF MF1-MF3 (n = 3) by group (n = 7). (B) UMAP plots of each MF and SS major and minor clones and occupation of cells in each group with bar graph (%) representative of percent of cells in each group. (C) Trajectory analysis and pseudotime (slingshot) analysis of SS cells (top) compared to MF cells (bottom). (D) Bubble plot of stemness score and exhaustion score colored by Shannon clonality index and sized by cell count of 7 groups. Bubble plot of single-cell ATAC-seq open chromatin regions and scRNA-seq gene expression patterns across phenotypes (stem-like, transition, effector, progenitor exhaustion, proliferation, mitochondrial activity, and T-regulatory) in SS (E) and MF (F). Color of each dot represents normalized average gene expression from high (red) to low (blue). Size of each dot represents the percentage of positive cells for each gene.

Figure 2.

TCR clonality patterns in SS and MF. (A) Quantification of number of clones, number of cells, and percent clone size in each sample for MF (MF1-MF3) and SS (SS1-SS4) major and minor clones from scRNA-seq and VDJ-seq. (B) Pie charts for composition of clones stratified by clone size, with color representing cells in different groups. (C) Shannon Clonality Index of TCR clones of MF and SS samples by group for major and minor clone T cells. UMAP representing the dominate clones for each MF (D) and SS (E) sample for CD4⁺CD26⁺ and CD4⁺CD26⁻ T cells detailing the distribution of the clone in each group. Clonality was calculated for groups ≥50 cells.

Figure 3.

Chromatin confirmation patterns in SS and MF. (A) UMAP plot of single cell ATAC-seq from CD4⁺CD26⁺ and CD4⁺CD26⁻ sorted T cells from peripheral blood of SS patients SS1-SS4 (n = 4) and MF patients MF3 (n = 1) by group (n = 5). (B) Individual UMAPs of each MF and SS CD4⁺CD26⁺ and CD4⁺CD26⁻ T cells, similar to panel A. (C) Violin plots of single cell ATAC-seq open chromatin regions of notable CTCL and T-cell effector genes such as SATB1, TCF7, CD69, ITGAE, PDCD1, TXB21, ZNF638, and ZEB2. (D) Violin plots of single cell ATAC-seq open chromatin regions of notable CTCL and T-cell effector genes such as IKZF1, IKZF2, and IKZF3. (E) Chromatin peaks near notable CTCL genes and T-cell genes such as SATB1, IKF2, ZEB1, RUNX3, and NOTCH1. Heat maps of group-specific (F) gene activity and (G) transcription factor motif enriched in chromatin peaks genes. Columns present different cell groups. Rows present (F) gene showing different activity across group and (G) transcription factor (TF) motif enriched in differentially accessible peak regions. Color in panel E presents gene-wise z-scaled normalized average activity. Color in panel F presents normalized enrichment of the TF motif enriched within the differentially accessible peak regions.

Figure 3.

Chromatin confirmation patterns in SS and MF. (A) UMAP plot of single cell ATAC-seq from CD4⁺CD26⁺ and CD4⁺CD26⁻ sorted T cells from peripheral blood of SS patients SS1-SS4 (n = 4) and MF patients MF3 (n = 1) by group (n = 5). (B) Individual UMAPs of each MF and SS CD4⁺CD26⁺ and CD4⁺CD26⁻ T cells, similar to panel A. (C) Violin plots of single cell ATAC-seq open chromatin regions of notable CTCL and T-cell effector genes such as SATB1, TCF7, CD69, ITGAE, PDCD1, TXB21, ZNF638, and ZEB2. (D) Violin plots of single cell ATAC-seq open chromatin regions of notable CTCL and T-cell effector genes such as IKZF1, IKZF2, and IKZF3. (E) Chromatin peaks near notable CTCL genes and T-cell genes such as SATB1, IKF2, ZEB1, RUNX3, and NOTCH1. Heat maps of group-specific (F) gene activity and (G) transcription factor motif enriched in chromatin peaks genes. Columns present different cell groups. Rows present (F) gene showing different activity across group and (G) transcription factor (TF) motif enriched in differentially accessible peak regions. Color in panel E presents gene-wise z-scaled normalized average activity. Color in panel F presents normalized enrichment of the TF motif enriched within the differentially accessible peak regions.

Figure 4.

Somatic mutations overlapping in HSCs and mature peripheral cells in SS. Upset plot of overlapping mutations across bone marrow HSCs, blood HSCs, CD26⁻ cells, CD26⁺ T cells, and monocytes with corresponding Venn Diagrams and combined density plots with line graphs of variant allele frequency (VAFs) of mutations overlapping between progenitors and T cells in 4 patient samples (A-D).

Figure 4.

Somatic mutations overlapping in HSCs and mature peripheral cells in SS. Upset plot of overlapping mutations across bone marrow HSCs, blood HSCs, CD26⁻ cells, CD26⁺ T cells, and monocytes with corresponding Venn Diagrams and combined density plots with line graphs of variant allele frequency (VAFs) of mutations overlapping between progenitors and T cells in 4 patient samples (A-D).

Figure 5.

Thymic egression of SS. (A) Schematic of recombination events in TCR rearrangement to generate signal joint TRECs and coding joint TRECs. (B) Quantification of sjTREC via real-time qPCR from naïve T cells from peripheral blood of CTCL patients that are CD4⁺CD26⁻ (n = 6) or CD4⁺CD26⁺ (n = 3) compared to age-matched healthy donors (n = 6), cord blood, benign ovarian tumor peripheral patient blood, and sample matched CD14⁺CD3⁻ monocytes (n = 6) using total DNA. Data are presented as mean ± standard error of the mean (SEM). ∗P < .05. Unpaired 2-tailed t test was used for calculating differences between means between experimental groups.

Figure 6.

Antigen presentation capacity in SS and MF. (A) Antigen pulsing of APCs with autologous Sézary peripheral blood CD4⁺CD26⁻CD7⁻ cells (n = 3) against no antigen, keratinocyte lysate (12 μg), or allogenic Sézary skin biopsy lysate (12 μg). Positive control for proliferation was CD3/CD28 stimulated T cells. Enzyme-linked immunosorbent assay (ELISA) of supernatant from antigen presented cells for human IL-4 with a 1:1 dilution. Values are represented as fold change over APCs and T-cell negative control condition. Experiment was done in triplicate. (B) Quantification of fold change over APC and T cells for CD69 activated live CD4⁺CD26⁻ T cells across keratinocyte, skin, APC and T cells only, and CD3/CD28 bead stimulated T cell conditions. Two-tailed Student t test: ∗P < .05; ∗∗P ≤ .01. (C) Conditions similar to panel A were performed in the peripheral blood MF samples CD4⁺CD26⁻/CD7⁻ cells (n = 3) for the quantification of fold change of CD69 activated T cells. Two-tailed Student t test was performed. (D) Histogram displaying the FACs staining for CD69 (n = 3). n.s., nonsignificance.