Dissection of cHL tumor microenvironment and the association of HRS-like cells with treatment response. (A) Schematic overview of the study. (B) Treatment regimens, response onset, and duration for tumors conducted scRNA-seq in this study. The color indicates DP or CDP therapy. The length of the bar shows PFS. The best clinical response for tumor biopsy is labeled. Patients with ongoing response are indicated by an arrow, and patients with progression or relapse are indicated by a cross. For UPN51, the biopsy sample before CDP therapy was a recurrent tumor, and the other 3 biopsy samples were collected at identical location. (C) Uniform manifold approximation and projection (UMAP) map of 185 791 cells in cHL. Colored by cell subtype. (D) Dot plots showing representative signature genes of cell subtypes. Color for z score scaled expression and size for detection rate. (E) Representative images of multiplex immunostaining results. The CD30 (red), DHRS2 (green), and PAX5 (pink) are used to highlight HRS-like cells. The CD30^–DHRS2⁺PAX5^dim+ cells are indicated by white arrows. At the time of sample collection, patient UPN33 was anti–PD-1 treatment naïve (P naive), and patient UPN19 had disease progression after prior anti–PD-1 therapy (P resist). (F) RNA velocities overlaid on principal component analysis showing the transition path of the HRS-like cells from CD30^– cells to CD30⁺ cells. Streamlines show the RNA velocity field, and dots are colored by CD30 expression status. (G) Volcano plot showing differentially expressed genes between CD30⁺ and CD30^– HRS-like cells. Red dots for significantly higher expression in the on-treatment samples; blue dots for significantly lower expression in the on-treatment samples. Representative genes are highlighted. (H) Box plots showing normalized counts of total HRS-like cells, CD30⁺ HRS-like cells, and CD30^– HRS-like cells for paired pretreatment (Pre) and on-treatment (On) samples. Adjusted P values and fold change values by negative binomial-generalized linear models were also shown. (I) CD30^– and CD30⁺ HRS cell proportions in a serial of tumors from the patient UPN51 who achieve PR after CDP treatment. (J) L1236-mCherry cells were stained with anti-CD30 antibody and incubated with peripheral blood mononuclear cells (PBMCs), which were stained with CellTrace eFluor dye, for 60 or 90 minutes at a ratio of 1:20. The percentages of conjugates formed by CD30⁺ or CD30^– L1236 with PBMCs (gating from CD30⁺ or CD30^– mCherry⁺ cells) were calculated. Results are pooled from 2 repeated experiments (n = 4 per group). Data are represented as mean ± standard error of the mean (SEM), by 2-tailed Student t test. (K) L1236-mCherry cells were pretreated with phosphate-buffered saline (PBS; con group), 100 nM chidamide (Chi group), 10 nM decitabine (DAC group), or 100 nM chidamide plus 10 nM decitabine (C + D group) for 3 days, followed by incubation with PBMCs-eFluor for 60 or 90 minutes at a ratio of 1:20. The percentages of conjugates formed by L1236 with PBMCs were calculated. Results are pooled from 2 repeated experiments (n = 4 per group). Data are represented as mean ± SEM, by 1-way analysis of variance (ANOVA). (L) Violin plots depicting the expression of major histocompatibility complex (MHC) class I/II genes in 5 groups. Comparisons were performed by the 2-sided Wilcoxon tests. NR, nonresponder; R, responder; scTCR-seq, single-cell TCR sequencing.