Gene-expression signature analysis of Notch-activated DLBCL. (A) Representative immunohistochemical images showing IL-33 staining of DLBCL samples with negative (left) and positive (right) expression of cytoplasmic IL-33. Samples were evaluated as positive for cytoplasmic IL-33 expression if the cytoplasm of 30% or more of tumor cells was stained for IL-33. Clinical characteristics and prognosis were compared between cytoplasmic IL-33–positive and –negative groups. (B) RNA-Seq gene-expression data derived from 624 DLBCL samples obtained from EGA. Notch-signaling activity was calculated as the geometric mean of the expression of 66 direct Notch target genes. Notch-signaling activity vs IL33 expression. (C) GSEA enrichment plot for DLBCL with high Notch-signaling activity (n = 312) vs that with low Notch-signaling activity (n = 312), and high IL33 expression (n = 312) vs low IL33 expression (n = 312), for genes upregulated in adult Treg cells in comparison with adult conventional T cells (GSE25087_TREG_VS_TCONV_ADULT_UP). (D) Intratumor immune cell activity was calculated as the geometric mean of the expression of immune metagenes. Notch-signaling activity and IL33 expression vs intratumor CD8⁺ T-cell activity (top), Treg cell activity (middle), and the log ratio of intratumor CD8⁺ T-cell activity to Treg cell activity (bottom). FDR, false discovery rate; FPKM, fragments per kilobase of exon per million fragments mapped; NES, normalized enrichment score; rho, Spearman rank correlation coefficient.