Abstract Background: Follicular lymphoma (FL) is a germinal center–derived B cell malignancy characterized by the clonal expansion of a dominant malignant B cell population. While prior studies have identified transcriptional subtypes across patients, most analyses have treated B cells within individual tumors as a transcriptionally uniform population. However, this assumption has not been systematically tested using high-resolution, clonotype-resolved transcriptomic data. The broader landscape of intratumoral B cells—including those not belonging to the dominant clone—remains poorly defined, and their roles in disease biology are not well understood. These unexpanded, intratumoral B (ITB) cells may include residual non-malignant populations, early-stage malignant cells, or immune-modulating cells with distinct roles in the tumor microenvironment. It is also unknown whether their presence varies across patients or correlates with clinical features or immune microenvironmental interactions. Methods: We performed integrated single-cell 5′ RNA sequencing and B cell receptor (BCR) repertoire sequencing on 7 newly diagnosed FL 1-3A fresh-frozen lymph node biopsies (Dasari et al., Blood Cancer Discov 2025). Using a high-confidence approach that incorporates heavy and light chain pairing, V and J gene usage, and CDR3 nucleotide junction identity, we classified B cells into two groups: (1) cells belonging to the dominant expanded clone (FL B cells) and (2) all other B cells with detectable and non-expanded BCRs (ITB). These clonal identities were then integrated with single-cell transcriptomic profiles using Seurat and Immcantation pipelines. Differential expression analysis was conducted using edgeR to compare FL B cells to ITB cells across all FL biopsies. Results: After quality filtering, a total of 44,637 single cells were retained across all 7 samples (minimum per sample: 3,847 cells), including malignant and non-malignant B cells as well as diverse tumor microenvironment populations. The proportion of B cells assigned to the dominant FL clone varied substantially across tumors, ranging from 22% to 89%, indicating that some tumors contain a substantial population of ITB cells. In one tumor, we identified two expanded malignant B cell clones comprising 40.6% and 34.4% of all cells. These clones exhibited non-overlapping V and J gene usage (IGHV3-11/IGHJ5 vs. IGHV3-72/IGHJ3), distinct CDR3 junctions, and different immunoglobulin isotypes (IGHG1 vs. IGM). BCL2 was expressed in most cells within both clones (Clone 1: 87.8%, Clone 2: 67.0%), with mean log-normalized expression levels of 1.79 and 0.95, respectively. Both clones formed distinct UMAP clusters and showed transcriptional divergence from ITBs, with 453 differentially expressed genes (DEGs) identified for Clone 1 and 98 for Clone 2 (adjusted p0.01, |log₂FC|1, min.pct≥0.25). Additionally, 748 DEGs were identified between the two clones. This evidence of clonal divergence within a single FL tumor reinforces the need to reconsider intratumoral homogeneity in FL and suggests that multiple malignant clones may sometimes coexist. When comparing FL clones to ITB cells across tumors we found that FL clones exhibited a transcriptional program enriched for proliferation and cell cycle regulators, including MKI67 (p0.005), E2F1 (p0.0001), CDKN2A (p0.0001), and CDKN2B (p0.01), as well as elevated expression of AICDA (p0.01), EZH2 (p0.0001), and PD-L1 (p0.05). As expected, the malignant cells also had higher expression of BCL2 (p0.01) and CD10/MME (p0.0001). In contrast, ITB cells demonstrated relatively higher expression of SELL (p0.0001) and TNFRSF14 (p0.05), which were significantly downregulated in the FL clone. Conclusions: Together, these findings challenge the prevailing notion that FL tumors are defined by a single homogeneous malignant B cell population. Instead, our data support a model in which multiple transcriptionally and functionally distinct B cell populations coexist within tumors, including dominant clones and a diverse population of ITB cells. This is, to our knowledge, the first study to comprehensively characterize these populations using integrated, lineage-aware single-cell multiomic profiling across FL patients. These insights may ultimately inform new strategies for understanding tumor evolution, immune evasion, and therapeutic targeting in follicular lymphoma.
Walker et al. (Mon,) studied this question.