Longitudinal single-cell profiling of the bone marrow heterogeneity identifies the T-cell niche supporting cancer persister cells in follicular lymphoma

Abstract Follicular lymphoma (FL) disease trajectory is characterized by a protracted preclinical stage, followed by iterative cycles of remission/relapse, suggesting that the elusive FL cancer precursor/persister cells (CPC) are not eradicated by current therapies and support early FL development and lymphoma recurrence. FL malignant B cells are dependent on a dynamic interplay with a heterogeneous tumor microenvironment (TME), dominated by amplified Treg, activated Tfh, and dysfunctional cytotoxic T cells. FL TME composition within invaded lymph nodes (LN) has been associated with clinical outcomes. However, neither CPC nor their specific supportive niches have been yet characterized, despite being the ultimate targets to cure FL. Compelling clinical evidence has identified the bone marrow (BM) as a permissive niche for FL CPC. Here, we analyze the BM T cell niche in low tumor burden FL patients treated by Rituximab monotherapy (FLIRT trial, NCT02303119), looking for the impact of malignant B cells on BM T cell profile and for the T-cell niche supporting drug-resistant persister cells. Paired BM aspirates were obtained from 19 low tumor-burden FL patients at diagnosis (M0) and in remission at one year (M12) of Rituximab monotherapy. BM aspirates from 5 age-matched healthy donors (HD) were incorporated as controls. We generated a single-cell dataset combining gene expression and BCR/TCR repertoires (10X Genomics) from purified B and T cells. Quantification of the t(14;18) frequency by QPCR revealed a systematic, yet variable, infiltration of FL BM at M0 and a dramatic depletion at M12, when few residual tumor cells could be detected and profiled. A total of 54,732 CD4 and 53,580 CD8 T cells were profiled. The BM T-cell landscape was highly heterogeneous across FL patients in contrast to the more uniform distribution in HD BM. Among the 15 T-cell clusters, 8 were differentially represented in FL BM at M0 vs HD BM with a decrease of cytotoxic CD8 T cells and naive CD4 T cells and an increase of IFN-responsive CD4 and CD8, Treg, Tfh-like, and memory CD8 T cells. Treg, IFN-responsive CD4, and CD8 EM were associated with a poorer clinical outcome, defined as a PFS<24 months and/or a time-to-next treatment (TTNT)<30 months (n=8/19 patients, P<0.05). Conversely, naive CD4 and naive CD8 T cells were associated with a favorable outcome. While exhausted CD8 T cells were not over-represented in FL BM, we identified a FL-specific subset of IL-10+ CD8 T cells co-expressing CD8 Treg markers. Furthermore, cytotoxic T cell transcriptional profile was characterized by a reduced expression of genes/pathways associated with T-cell activation and cytotoxicity, while the TGFb pathway was enriched, collectively highlighting a shift towards local immune suppression. At M12, IFN-responsive CD4, Treg, naive CD4 and CD8 T cells returned to levels similar to those found in HD BM, suggesting that their alteration at M0 was dependent on tumor B cell infiltration. However, M0 and M12 FL BM Treg retained a similar activated TNFRSF9hi Treg signature, which was associated with a poor prognosis when scored on bulk RNAseq of FLIRT FL LN biopsies at diagnosis (P=0.0048, n=122). Furthermore, the defective cytotoxic CD8 response persisted both quantitatively and qualitatively upon B-cell depletion. Finally, the Tfh-like cluster remained high at M12 in FL BM, regardless of the presence of residual tumor B cells. Analysis of BM malignant B cell heterogeneity using an unsupervised metaclustering approach identified 7 metaclusters (Mcl) co-occurring across FL patients and co-opting distinct gene expression programs, resembling the B cell developmental hierarchy in the LN from dark zone GC-like, light zone (LZ) GC-like, and memory-like FL B cells. Interactome analysis revealed a preferential interaction between BM Tfh-like cells and BM LZ GC-like tumor B cells. Interestingly, M12 residual tumor B cells were enriched for LZ GC-like Mcl, in agreement with the persistence of Tfh-like T cells.Our data describe the T-cell landscape within FL BM TME, unveiling a profound enrichment of regulatory T cell subsets and Tfh-like cells, alongside a defective cytotoxic immune response. Following B cell depletion using a B-cell targeted monotherapy, the tumor supportive T-cell niche persists within the BM. This preserved niche may support the survival of rare residual GC-like CPC and contribute to subsequent relapses, thereby identifying a new therapeutic vulnerability in FL.