Dynamic crosstalk between CLL cells and lymph node fibroblasts promotes a pro-tumor microenvironment

Abstract

Abstract Chronic lymphocytic leukemia (CLL) relies heavily on microenvironmental support within secondary lymphoid organs, especially lymph nodes (LNs). Within LNs, proliferation centers provide critical survival and growth signals that drive leukemic expansion and immune evasion. Recentimaging-based spatial proteomic analysis of CLL patient LNs revealed a disrupted fibroblastic reticular cell (FRC) network, characterized by loss of PDPN, CD21, and CD35, alongside the presence of CCL19highCCL21low LN fibroblasts that interact with MYChighKi-67high CLL cells in proliferation centers. In parallel, scRNA-seq of these LNs identified distinct cellular clusters and significant signaling between stromal cells and cycling CLL cells, supporting dynamic crosstalk between FRCs and CLL. Thus, we focus here on FRCs, the key organizers of LN architecture and immune regulation, to study their role in supporting CLL disease progression. We previously reported that the size of cycling CLL cells within LNs correlates with poor clinical outcomes. Using 2H2O labeling to track dividing cells, we identified the CXCR4DimCD5Bright “proliferative fraction” PF as enriched in newly born cells, in contrast to the quiescent CXCR4BrightCD5Dim “resting fraction” (RF). Notably, PF cells or CLL cells activated by anti-IgM and IL-4 secrete significantly higher levels of lymphotoxin, which stimulates FRCs and enhances CCL19 production, suggesting that cycling CLL cells can directly reprogram LN fibroblasts. Indeed, in co-cultures of human LN fibroblasts (HLFs) with distinct CLL B-cell fractions, only PF or activated CLL cells, but not RF cells, induced characteristic cancer associated fibroblast (CAF)-like changes, including cellular stretching, traction, and the emergence of filopodia-like extensions that may facilitate malignant B-cell infiltration. We next examined the role of lymphotoxin beta receptor (LTβR) signaling in FRCs by treating HLFs with soluble lymphotoxin for 3 days in a dose-dependent manner. In both 2-D and 3-D systems, lymphotoxin significantly promoted FRC proliferation (~1.5-fold). In the 3-D cultures, lymphotoxin increased PDPN expression, enhanced secretion of key chemokines, including CCL19 and CXCL12, and reduced PD-L1. Additionally, lymphotoxin stimulation induced CAF-like morphological features in FRCs. These phenotypic and morphological changes were absent in the 2-D system, highlighting the limitations of traditional monolayer cultures in modeling lymphoid stromal responses, and support the idea that cycling CLL cells in LN proliferation centers produce lymphotoxin that functionally reprograms FRCs via LTβR signaling. Finally, we aim to identify therapeutic targets that mediate FRC-CLL interactions. The scRNA-seq of CLL patient LNs described above indicates that CLECL1⁺ but not CLECL1⁻ CLL cells are enriched for CCR7 expression and lymphotoxin production. CLECL1 is a costimulatory molecule known to activate T cells and monocytes. Exposure of HLFs to rCLECL1 or co-culture HLFs with activated CLECL1⁺ CLL cells induced fibroblast morphological changes, as well as secretion of CCL19 and CXCL12. Enrichment of CLECL1⁺ CLL cells also promotes the formation of nurse-like cells, indicating a role for CLECL1 in both stromal and accessory cell activation. To evaluate CLECL1 as a therapeutic target, we applied IgG monoclonal antibodies (mAbs) against the external domain of human CLECL1. These anti-CLECL1 mAbs block NLC and FRC activation. Supporting this, patient-derived xenografts demonstrated expansion of CLECL1⁺ B cells and reprogrammed fibroblasts. Depletion of CLECL1⁺ B cells in this model led to reduced Th2 cells and impaired CLL cell growth. Collectively, these studies demonstrate that FRCs are dynamic participants in the CLL tumor microenvironment, actively reprogrammed by cycling CLL cells through lymphotoxin and CLECL1-mediated signaling. This bidirectional crosstalk promotes a CAF-like fibroblast phenotype, marked by altered morphology, enhanced chemokine secretion, and diminished immune-regulatory signals, ultimately fostering disease progression. Our findings highlight CLECL1⁺ CLL cells as a functionally distinct subset capable of driving FRC activation and show that therapeutic targeting of CLECL1 can disrupt these pathogenic interactions. Together, this work uncovers key mechanisms by which CLL hijacks lymphoid stromal networks and highlights novel stromal-directed targets for disrupting microenvironmental support in CLL.