Fig. 2: The clinical impact of TLS maturation on development of antitumor immunity in HGSOC.

A Supervised hierarchical clustering of 53 tumor samples of HGSOC patients (Study Cohort 1) with TLS development (no TLSs, Cluster 1, n = 23), only early TLS (eTLS, Cluster 2, n = 18), early and mature TLS (eTLS+mTLS, Cluster 3, n = 12) based on the expression of 100 genes classified into clusters related to B cells, cytotoxicity, dendritic cells (DCs), immune cells, immunosuppression, natural killers (NK) cells, T cell activation, T_EM, T_H1 and T_H2 signatures as determined on RNA sequencing data from Study cohort 1. B Gene expression signature associated with B cells, T cells, CD8⁺ T cells as determined by MCP counter on RNAseq data and relative gene expression levels of PDCD1, HAVCR2, CTLA4 as determined on RNAseq data across patients (n = 53; Study cohort 1) separated into 3 clusters (clusters determined by immunofluorescence in (A), CL1: n = 23; CL2: n = 18; CL3: n = 12). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. ns not significant. C Unsupervised hierarchical clustering of gene signatures related to immune populations (orange), immune functions (green) and immune phenotype (purple) in tumor samples of 304 HGSOC patients from TCGA public database (Study cohort 4). D Representative images of double immunohistochemistry for CD3 and FoxP3, DC-LAMP and CD20 cells, single IHC for CD8, NKp46, CTLA-4, PD1, LAG-3 and double immunofluorescence (IF) staining of CD8 GZMB cells. Scale bar, 100 µm. E Unsupervised hierarchical clustering of HGSOC patients (n = 115; Study Cohort 1) based on frequency of TLS and densities of CD8⁺ T cells, DC-LAMP⁺ DCs, NKp46⁺ NK cells, FoxP3⁺ cell, PD1⁺, CTLA-4⁺ and LAG-3⁺ cells as determined by immunostaining. F Density of CD8⁺ (CL1: n = 44; CL2: n = 45; CL3: n = 17), tumor core CD8⁺ (CL1: n = 21; CL2: n = 24; CL3: n = 10) and GZMB⁺CD8⁺ cells (CL1: n = 24; CL2: n = 32; CL3: n = 9) in tumor samples of HGSOC patients (Study Cohort 1) separated into 3 clusters (CL1, no TLS development; CL2, only eTLS development; CL3, both eTLS and mTLS development), as determined by immunostaining. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. p values are indicated. G, H Relapse-free survival (RFS) and overall survival (OS) of 209 HGSOC patients (Study Cohort 1 and 2; Supplementary Table 1) based on median stratification of total TLS (G) and based on stratification into 3 clusters (CL1, no TLS development, n = 87; CL2, only eTLS development, n = 86; CL3, both eTLS and mTLS development, n = 36). Survival curves were estimated by the Kaplan-Meier method, and differences between groups were evaluated using log-rank test. Number of patients at risk and p values are reported. *p < 0.01; **p < 0.001; ***p < 0.0001. Source data and exact p values are provided as a Source Data file.