The Follicular Lymphoma and Chronic Lymphocytic Leukemia Proliferative Microenvironment at Single-Cell Resolution
Authors: Ferreira Antonio , Wang Shumei , Poluben Larysa , Brandstadter Joshua D , Perkey Eric , Sotirakos Steven , Drew Mai , Pan Li , Lemieux Madeleine , Dorfman David , Shoji Brent , Maillard Ivan , Blacklow Steve , Aster Jon C. 
Published: December 2024

Introduction
Lymph nodes (LNs) are secondary lymphoid organs where lymphocytes interact with antigen presenting cells to initiate adaptive immune responses within microenvironments established by resident stromal cells. LNs are also the major sites of follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) growth. These two B cell neoplasms alter the stromal architecture of LNs in highly stereotypical ways that are thought to be critical to facilitate their growth and survival.
Methods
To investigate the tumor microenvironment (TME) in FL and CLL, we developed a comprehensive single-cell RNA sequencing (scRNA-seq) pipeline to analyze all resident LN cells. This method involved independently sorting CD45+ (immune/hematopoietic) and CD45- cells (non-hematopoietic stroma), allowing the detailed examination of the gene expression profiles of the immune and non-hematopoietic stromal compartments. We analyzed over 82,000 immune and stromal cells derived from FL, CLL and immune reactive control LNs (n=3 for each condition). Tumor and non-tumor B lineage cells were differentiated using mRNA copy number variations identified by InferCNV. Additionally, bioinformatic analyses were conducted to predict ligand-receptor interactions between neoplastic cells and their respective LN TMEs, which were subsequently validated using multiplex fluorescence confocal microscopy on FFPE samples from FL (n=9) and CLL (n=13) patients, compared to control reactive LNs (n=4).
Results
Our findings revealed that the proliferation of FL and CLL cells within specialized niches is associated with a transient upregulation of MYC, subsequent downregulation of which may act to limit the growth potential of these indolent neoplasms. Proliferating FL cells within neoplastic follicles co-localized with follicular dendritic cells, whereas proliferating CLL cells were spatially associated with a distinct set of fibroblasts expressing CCL19 that localized to proliferation centers. We named this previously undescribed population CLL Proliferation-Associated (CPA) fibroblasts. Additionally, bioinformatic analysis paired with multiplex fluorescence microscopy validation identified interacting sets of ligand-receptor pairs established between proliferating neoplastic B cells with their respective TMEs, such as immune cells and stromal fibroblasts. Among the interactions detected were ligand-receptor pairs linked to immune cell regulation and tumor immune evasion such as CD74-MIF, CD55-CD97 and GAL9(Galectin-9)-CD44. We also detected spatial segregation between MYC+ proliferating FL cells and CD8 cytotoxic T cells, a pattern not observed with MYC+ CLL cells, suggesting differential abilities of these neoplasms to trigger a proliferative anti-tumor immune microenvironment.
Conclusion
In conclusion, we delineated specific proliferative niches for FL and CLL characterized by MYC upregulation and identified a novel population of CCL19+ CPA fibroblasts enriched at CLL proliferation centers, suggesting a central role in CLL proliferation. The distinct ligand-receptor interactions in these microenvironments indicate a role for immune evasion in the pathogenesis of these neoplasms. Overall, these insights provide a roadmap for potential therapeutic interventions targeting the vulnerabilities of FL and CLL within their respective TMEs.