HDAC inhibitor treatment affect some malignant subpopulations, but not all. (A) Coexpression of surface marker expression within malignant cells from a patient with SS (SS8) visualized by t-SNE plots colored by fluorescence intensity of the indicated markers or by automated clustering using the PhenoGraph algorithm showing all (left) or a reduced number of clusters (right). (B-G) Changes in the malignant subpopulations after treatment with increasing concentrations of 2 HDAC inhibitors. (B-D) Romidepsin or (E-G) SAHA colored by reduced PhenoGraph clusters. (B,E) Visualized by changes in t-SNE plots of clustered cells. (C-D,F-G) Visualized by stacked bar plots of (C,F) cluster frequency or (D,G) total cell counts. (H) Single-cell heat maps of malignant T cells treated with increasing concentrations of romidepsin. Rows are distributed by reduced PhenoGraph clusters (left) and colored by fluorescence intensity of the indicated markers. Violin plots (top) display the overall expression range of the indicated markers within the total malignant population. (I) Normalized quantification of the population diversity within the malignant population of 6 patients with SS after treatment with increasing concentrations of romidepsin or SAHA, based on distribution among PhenoGraph clusters, using different diversity indices (Shannon, Simpson, and inverse Simpson indices and Fisher’s α diversity). Diversity indices were normalized to the diversity of the untreated sample from each patient. Bars depict mean percentage ± standard error of mean of 6 patients with SS (n = 6). Note that cells from SS5 were treated with slightly different concentrations (see “Methods”).